Cytochrome P-450 monooxygenases

ABSTRACT

New cytochrome P-450 dependent monooxygenases and DNA molecules encoding these monooxygenases are provided, which are able to catalyze the biosynthetic pathway from amino acids to their corresponding cyanohydrins, the presursors of the cyanogenic glycosides, or to glucosinolates. Moreover, the invention provides methods for obtaining DNA molecules according to the invention and methods for obtaining transgenic plants resistant to insects, acarids, or nematodes or plants with improved nutritive value.

This application is a § 371 of PCT/EP94/03938, filed on Nov. 28, 1994,and published on Jun. 15, 1995, as WO 95/16041, which claims priority ofEuropean Patent Application No. 93810860.2, filed on Dec. 8, 1993.

The present invention relates to genetic engineering in plants usingrecombinant DNA technology in general and to enzymes involved in thebiosynthesis of cyanogenic glycosides and glucosinolates and genesencoding these enzymes in particular. The proteins and genes accordingto the invention can be used to improve the nutritive value or pestresistance of plants.

Cyanogenic glycosides constitute secondary plant metabolites in morethan 2000 plant species. In some instances they are the source of HCNwhich can render a plant toxic if it is taken as food. For example thetubers of the cyanogenic crop cassava (Manihot esculenta) constitute animportant staple food in tropical areas. However, the cyanogenicglycosides present in the tubers may cause cyanide poisoning in humansdue to insufficiently processed cassava products. Other plant specieswhose enzymatic production of HCN accounts for their potential toxicityif taken in excess as food or used as animal feed include white clover(Trifolium repens), sorghum (Sorghum bicolor), linen flax (Linumusitatissimum), triglochinin (Triglochin maritima), lima beans(Phaseolus lunatus), almonds (Amygdalus) and seeds of apricot (Prunus),cherries and apple (Malus). The toxic properties could be reduced byblocking the biosynthesis of cyanogenic glycosides in these plants.

The primary precursors of the naturally occuring cyanogenic glycosidesare restricted to the five hydrophobic protein amino acids valine,leucine, isoleucine, phenylalanine and tyrosine and to a singlenon-protein amino acid, cyclopentenylglycine. These amino acids areconverted in a series of reactions to cyanohydrins which are ultimatelylinked to a sugar residue. Amygdalin for example constitutes theO-β-gentiobioside and prunasin the O-β-glucoside of (R)-mandelonitrile.Another example of cyanogenic glycosides having aromatic aglycones isthe epimeric pair of the cyanogenic glycosides dhurrin and taxiphyllinwhich are to be found in the genus Sorghum and Taxus, respectively.p-Hydroxymandelo-nitrile for example is converted into dhurrin by aUDPG-glycosyltransferase. Similiar glycosyltransferases are believed tobe present in most plants. Vicianin and lucumin are further examples fordisaccharide derivatives similiar to amygdalin. Sambunigrin contains(S)-mandelonitrile as its aglycone and is therefore epimeric toprunasin.

Examples of cyanogenic glycosides having aliphatic aglycones arelinamarin and lotaustralin found in clover, linen flax, cassava andbeans. A detailed review on cyanogenic glycosides and their biosynthesiscan be found in Conn, Naturwissenschaften 66:28-34, 1979, hereinincorporated by reference.

The biosynthetic pathway for the cyanogenic glucoside dhurrin derivedfrom tyrosine has been extensively studied (Halkier et al, `Cyanogenicglucosides: the biosynthetic pathway and the enzyme system involved` in:`Cyanide compounds in biology`, Wiley Chichester (Ciba FoundationSymposium 140), pages 49-66, 1988; Halkier and Moller, Plant Physiol.90:1552-1559, 1989; Halkier et al, The J. of Biol. Chem.264:19487-19494, 1989; Halkier and Moller, Plant Physiol. 96:10-17,1990, Halkier and Moller, The J. of Biol. Chem. 265:21114-21121, 1990;Halkier et al, Proc. Natl. Acad. Sci. USA 88:487-491, 1991; Sibbesen etal, in: `Biochemistry and Biophysics of cytochrome P-450. Structure andFunction, Biotechnological and Ecological Aspects`, Archakov, A. I.(ed.), 1991, Koch et al, 8th Int. Conf. on Cytochrome P450, AbstractPII.053; and Sibbesen et al, 8th Int. Conf. on Cytochrome P450, AbstractPII.016). It has been found that L-Tyrosine is converted top-hydroxy-mandelonitrile, the precursor of dhurrin withN-hydroxytyrosine and supposedly N,N-dihydroxytyrosine,2-nitroso-3-(p-hydroxyphenyl)propionic acid, (E)- and(Z)-p-hydroxyphenylacetaldehyd oxime, and p-hydroxyphenylacetonitrile askey intermediates. Two monooxygenases dependent on cytochrome P-450 havebeen reported to be involved in this pathway. A similiar pathway alsoinvolving cytochrome P-450 dependent monooxygenases has beendemonstrated for the synthesis of linamarin and lotaustrain from valineand isoleucine respectively in cassava (Koch et al, Archives ofBiochemistry and Biophysics, 292:141-150, 1992).

It has now surprisingly been found that the complex pathway fromL-tyrosine to p-hydroxy-mandelonitrile summarized above can bereconstituted by two enzymes only, which turn out to be identical to thecytochrome P-450 dependent monooxygenases. This result is verysurprising given the high degree of complexity of the pathway reflectedby its numerous intermediates. Thus the two cytochrome P-450monooxygenases are multifunctional. A first enzyme, designatedP-450_(I), converts the parent amino acid to the oxime. A second enzyme,designated P-450_(II), converts the oxime to the cyanohydrin.Multifunctional cytochrome P-450 enzymes have not previously been foundand described in plants.

Glucosinolates are hydrophilic, non-volatile thioglycosides found withinseveral orders of dicotyledoneous angiosperms (Cronquist, `The Evolutionand Classification of Flowering Plants, New York Botanical Garden,Bronx, 1988). Of greatest economic significance is their presence in allmembers of the Brassicaceae (order of Capparales), whose many cultivarshave for centuries provided mankind with a source of condiments,relishes, salad crops and vegetables as well as fodders and foragecrops. More recently, rape (especially Brassica napus and Brassicacampestris) has emerged as a major oil seed of commerce. About 100different glucosinolates are known possessing the same general structurebut differing in the nature of the side chain. Glucosinolates are formedfrom protein amino acids either directly or after a single or multiplechain extension (Underhill et al, Biochem. Soc. Symp. 38:303-326, 1973).N-hydroxy amino acids and aldoximes which have been identified asintermediates in the biosynthesis of cyanogenic glycosides also serve asefficient precursors for the biosynthesis of glucosinolates (Kindl etal, Phytochemistry 7:745-756, 1968; Matsuo et al, Phytochemistry11:697-701, 1972; Underhill, Eur. J. Biochem. 2:61-63, 1967).

It has now surprisingly been found that the cytochrome P-450_(I)involved in cyanogenic glycoside synthesis is very similiar to thecorresponding biosynthetic enzyme in glucosinolate synthesis.

The reduction of the complex biosynthetic pathway for cyanohydrinsdescribed above to the catalytic activity of only two enzymes,cytochrome P-450_(I) and P-450_(II), allows the introduction of thebiosynthetic pathway of dhurrin into plants, which plants in theirwildtype phenotype do not normally produce cyanogenic glycosides. Bytransfection of gene constructs coding for one or both of the twocytochrome P-450 monooxygenases it will be possible to eitherreconstitute or newly establish a biosynthetic pathway for cyanogenicglycosides. It is therefore an object of the present invention toprovide genes coding for cytochrome P-450 monooxygenases active in thebiosynthesis of cyanogenic glycosides.

The introduction of a biosynthetic pathway for cyanogenic glycosidesinto plants by methods known in the art, which in their wildtypephenotype do not express these glycosides is of great interest. This isdue to the surprising finding of the present invention that cyanogenicglycosides can be toxic to insects, acarids, and nematodes. Therefore,the introduction or reconstitution of a biosynthetic pathway forcyanogenic glycosides in plants or certain plant tissues will allow torender plants toxic to insects, acarids or nematodes and thus help toreduce the damage to the crop by pests. In combination with otherinsecticidal principles such as Bacillus thuringiensis endotoxins thedamage to the crop by pests could be even further reduced.

Alternatively, the sequences of the genes encoding the monooxygenasesaccording to the invention can be used to design DNA plasmids which upontransfection into a plant containing cyanogenic glycosides such ascassava, sorghum or barley eliminate cyanogenic glycosides normallyproduced in wildtype plants. This can be achieved by expression ofantisense or sense RNA or of ribozymes as described in EP-458 367 A1,EP-240 208-A2, U.S. Pat. No. 5,231,020, WO 89/05852, and WO 90/11682which RNA inhibits the expression of monooxygenases according to theinvention. This is of great interest as in spite of numerous efforts ithas not been possible through traditional plant breeding to completelyremove the cyanogenic glycosides from for example cassava and sorghum.On the other hand it has been shown that elevated amounts of cyanogenicglycosides in the epidermal cells of barley cultivars confer increasedsensitivity to attack by the mildew fungus Erysiphe graminis(Pourmohensi, PhD thesis, Gottingen, 1989; Ibenthal et al, Angew. Bot.67:97-106, 1993). A similiar effect has been observed in the cyanogenicrubber tree Hevea brasiliensis upon attack by the fungus Microcyclusulei (Lieberei et al, Plant Phys. 90:3-36, 1989) and with flax attackedby Colletotrichum lini (Ludtke et al, Biochem. Z. 324:433-442, 1953). Inthese instances the quantitative resistance of the plants stipulatedabove and of other plants, where cyanogenic glycosides confer increasedsensitivity to attack by microorganisms, can be increased by preventingthe production of cyanogenic glycosides in such plants. In barley, thecyanogenic glycosides are located in the epidermal cells. The antisene,sense or ribozyme constructs are therefore preferably but but notnecessarily combined with an epidermis specific promoter.

The presence of even minor amounts of cyanogenic glycosides in plantsmay also cause nutritional problems due to generation of unwantedcarcinogens as demonstrated in barley. Barley malt for example containslow amounts of the cyanogenic glucoside epiheterodendrin which in thecause of production of grain-based spirits can be converted toethylcarbamate which is considered to be a carcinogen. Attempts arebeing made to introduce mandatory maximum allowable concentrations ofethylcarbamate in fermented food, beverages and spirits (Food ChemicalNews 29:33.35, 1988).

Plants containing cyanogenic glycosides typically contain only a singlecyanogenic glycoside or just a few. In certain cases it is of interestto alter the cyanogenic glycoside profile of a plant. Since cytochromeP-450_(II) shows broad substrate specificity this enzyme typicallyconverts the aldoxime produced by cytochrome P-450_(I) into thecorresponding cyanohydrin. Alteration of the chemical identity ofcyanogenic glycosides produced in a specific plant can thus beaccomplished by transforming a plant with an additional gene encoding anexpressible cytochrome P-450_(I) monooxygenase with a substratespecificity different from the naturally occuring enzyme.

The present invention relates primarily to a DNA molecule coding for acytochrome P-450 monooxygenase, which catalyzes the conversion of anamino acid to the corresponding N-hydroxyamino acid and the oximederived from this N-hydroxyamino acid. Preferably the inventivemonooxygenase catalyzes the conversion of an amino acid selected fromthe group consisting of tyrosine, phenylalanine, tryptophan, valine,leucine, isoleucine and cyclopentenylglycine or an amino acid selectedfrom the group consisting of L-tyrosine, L-valine and L-isoleucine.Additionally the present invention relates to a DNA molecule coding fora cytochrome P-450 monooxygenase, which monooxygenase catalyzes theconversion of said oxime to a nitrile and the conversion of said nitrileto the corresponding cyanohydrin. The DNA molecules according to theinvention either correspond to naturally occuring genes or to functionalhomologues thereof which are the result of mutation, deletion,truncation, etc. but still encode cytochrome P-450 monooxygenases, whicheither catalyze the conversion of an amino acid to the correspondingN-hydroxyamino acid and the oxime derived from this N-hydroxyamino acid,or the conversion of said oxime to a nitrile and the subsequentconversion of said nitrile to the corresponding cyanohydrin. Bothmonooxygenases are able to catalyze more than one reaction of thebiosynthetic pathway of cyanogenic glycosides but preferably contain asingle catalytic center. The monooxygenase cytochrome P-450_(I)converting the parent amino acid is also involved in glucosinolatebiosynthesis. Because cytochrome P-450_(I) determines the substratespecificity and thus the type of glucosinolates produced and becausecytochrome P-450_(I) constitutes the rate limiting step, the principlesalready described above for cyanogenic glucosides can also be used todown- or up-regulate the synthesis of glucosinolates in glucosinolateproducing plants and to alter the compositon of glucosinolates produced.

The inventive DNA molecule encoding cytochrome P-450_(I) is obtainablefrom plants which produce cyanogenic glycosides and glucosinolates.These plants include but are not limited to plants selected from thegroup consisting of the species Sorghum, Trifolium, Linum, Taxus,Triglochin, Mannihot, Amygdalus and Prunus as well as cruciferousplants. In a preferred embodiment of the invention the DNA molecule isobtained from Sorghum bicolor (L.) Moench or Manihot esculenta Crantz.The sequence similarity between cytochrome P-450_(I) monooxygenases fromdifferent plants producing cyanogenic glycosides or glucosinolates isevidenced by the specific cross-reactivity of antibodies preparedagainst cytochrome P-450_(TYR) isolated from sorghum, with thecorresponding cytochrome P-450 enzyme in cassava and with thecorresponding enzyme in the glucosinolate producing plant Tropaeolummajus. Southern blotting using the cDNA clone encoding cytochromeP-450_(TYR) shows specific and strong hybridization to genomic DNAisolated from cassava, Tropaeolum majus, and rape. Of all approximately250 known published sequences for cytochrome P-450 enzymes, cytochromeP-450_(TYR) shows the highest sequence similarity to the petunia3'5'-flavonoid hydroxylase (30,8%) and 28% sequence similiarity toCYP1A2 from rabbit. The group of cytochrome P-450_(I) monooxygenasesfunctionally characterized by catalyzing the conversion of an amino acidto the corresponding aldoxime can thus be defined as cytochrome P-450enzymes the amino acid sequence of which exhibits a 32% or highersequence similarity and preferably a 40% or higher sequence similarityto that of cytochrome P-450_(TYR). Cytochrome P-450 gene proteinfamilies are defined as having less than 40% amino acid identity to acytochrome P-450 protein from any other family. Consequently, cytochromeP-450_(TYR) belongs to a new P-450 protein family.

The inventive DNA molecule encoding cytochrome P-450_(II) is obtainablefrom plants which produce cyanogenic glycosides. In a preferredembodiment of the invention the DNA molecule is obtained from Sorghumbicolor (L.) Moench or Manihot esculenta Crantz. The enzyme isolatedfrom Sorghum bicolor (L.) Moench is designated cytochrome P-450_(Ox).The catalytic properties of this enzyme resembles those of a cytochromeP-450 activity reported in microsomes from rat liver (DeMaster et al, J.Org. Chem. 5074-5075, 1992) which has neither been isolated nor furthercharacterized. A characteristic of cytochrome P-450_(Ox) and of othermembers belonging to the cytochrome P-450_(Ox) family is thatdehydration of the oxime to the corresponding nitrile is dependent onthe presence of NADPH but that this dependence can be overcome by theaddition of sodium dithionite or other reductants. Cytochrome P-450enzymes able to convert aldoximes into cyanohydrins might be present inmost living organisms.

For the purposes of gene manipulation using recombinant DNA technologythe DNA molecule according to the invention may in addition to the genecoding for the monooxygenase comprise DNA which allows for examplereplication and selection of the inventive DNA in microorganisms such asE. coli, Bacillus, Agrobacterium, Streptomyces or yeast. It may alsocomprise DNA which allows the monooxygenase genes to be expressed andselected in homologous or heterologous plants. Such sequences comprisebut are not limited to genes whose codon usage has been adapted to thecodon usage of the heterologous plant as described in WO 93/07278; togenes conferring resistance to neomycin, kanamycin, methotrexate,hygromycin, bleomycin, streptomycin, or gentamycin, toaminoethylcystein, glyophosphate, sulfonylurea, or phosphinotricin; toscorable marker genes such as galactosidase; to its natural promoter andtranscription termination signals; to promoter elements such as the 35Sand 19S CaMV promoters, or tissue specific plant promoters such aspromoters specific for root (described for example in EP-452 269-A2, WO91/13992, U.S. Pat. No. 5,023,179), green leaves such as the maizephosphoenol pyruvate carboxylase (PEPC), pith or pollen (described forexample in WO 93/07278), or inducible plant promoters (EP 332 104); andto heterologous transcription termination signals.

The present invention also relates to monooxygenases which catalyze theconversion of an amino acid preferably selected from the groupconsisting of tyrosine, phenylalanine, tryptophan, valine, leucine,isoleucine and cyclopentenylglycine to the corresponding N-hydroxyaminoacid and the oxime derived from this N-hydroxyamino acid (cytochromeP-450_(I)); or the conversion of said oxime to a nitrile and theconversion of said nitrile to the corresponding cyanohydrine (cytochromeP-450_(II)). In a preferred embodiment of the invention themonooxygenases are purified and can be used to establish monoclonal orpolyclonal antibodies which specifically bind to the monooxygenases.

In another preferred embodiment of the invention the cytochromeP-450_(II) monooxygenase can be isolated from Sorghum, has a molecularweight of 51 kD as determined by SDS-PAGE and comprises the N-terminalsequence

    MDLADIPKQQRLMAGNALVV                                       (SEQ ID NO: 12).

For other cytochrome P-450_(II) enzymes, the N-terminal sequences may bedifferent.

Optionally, a P-450_(II) monooxygenase might also comprise one of thefollowing sequences:

    --ARLAEIFATII--                                            (SEQ ID NO:13)

    --EDFTVTTK--                                               (SEQ ID NO: 14)

    --QYAALGSVFTVPII--                                         (SEQ ID NO: 15)

    --XXPFPI--(SEQ ID NO: 16).

Another embodiment of the present invention deals with a method for thepreparation of cDNA coding for a cytochrome P-450 monooxygenase, whicheither catalyzes the conversion of an amino acid preferably selectedfrom the group consisting of tyrosine, phenylalanine, tryptophan,valine, leucine, isoleucine and cyclopentenylglycine, to thecorresponding N-hydroxyamino acid and the oxime derived from thisN-hydroxyamino acid (cytochrome P-450_(I)); or the conversion of saidoxime to a nitrile and the conversion of said nitrile to thecorresponding cyanohydrin (cytochrome P-450_(II)); comprising

(a) isolating and solubilizing microsomes from plant tissue producingcyanogenic glycosides or glucosinolates,

(b) purifying the cytochrome P-450 monooxygenase,

(c) raising antibodies against the purified monooxygenase,

(d) probing a cDNA expression library of plant tissue producingcyanogenic glycosides or glycosinolates with said antibody, and

(e) isolating clones which express the monooxygenase.

Microsomes can be isolated from plant tissues which show a high activityof the enzyme system responsible for biosynthesis of the cyanogenicglycosides. These tissues may be different from plant species to plantspecies. A preferred source of microsomes are freshly isolated shootsharvested 1 to 20 days, preferably 2 to 10 days and most preferably 2 to4 days after germination. Etiolated seedlings are preferred from plantproducing cyanogenic glycosides but light grown seedlings may also beused. Following isolation the microsomes are solubilized in buffercontaining one or more detergents. Preferred detergents are RENEX 690(J. Lorentzen A/S, Kvistgard, Denmark), reduced Triton X-100 (RIX-100)and CHAPS.

The cytochrome P-450 monooxygenases can be purified applying standardtechniques for protein purification such as ultracentrifugation,fractionated precipitation, dialysis, SDS-PAGE and columnchromatography. Possible columns comprise but are not limited to ionexchange columns such as DEAE Sepharose, Reactive dye columns such asCibacron yellow 3 agarose, Cibacron blue agarose and Reactive red 120agarose, and gel filtration columns such as Sephacryl S-1000. Thecytochrome P-450 content of the individual fractions can be determinedfrom carbon monoxide difference spectra.

The purified proteins can be used to elicit antibodies in for examplemice, goats, sheeps, rabbits or chickens upon injection. 5 to 50 μg ofprotein are injected several times during approximately 14 dayintervals. In a preferred embodiment of the invention 10 to 20 μg areinjected 2 to 6 times in 14 day intervals. Injections can be done in thepresence or absence of adjuvants. Inmunoglobulins are purified from theantisera and spleens can be used for hybridoma fusion as described inHarlow and Lane, `Antibodies: A Laboratory Manual`, Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y., 1988, herein incorporated byreference. Antibodies specifically binding to a cytochrome P-450monooxygenase can also be used in plant breeding to detect plantsproducing altered amounts of cytochrome P-450 monooxygenases and thusaltered amounts of cyanogenic glycosides.

The methods for the preparation of plant tissue cDNA libraries areextensively described in Sambrook et al, Molecular cloning: A laboratorymanual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,1989, the essential parts of which regarding preparation of cDNAlibraries are herein incorporated by reference. PolyA⁺ RNA is isolatedfrom plant tissue which shows a high activity of the enzyme systemresponsible for biosynthesis of the cyanogenic glycosides orglucosinolates. These tissues may be different from plant species toplant species. A preferred tissue for polyA⁺ RNA isolation is the tissueof freshly isolated shoots harvested 1 to 20 days, preferably 2 to 10days and most preferably 2 to 4 days after germination. When cDNAlibraries are made from glucosinolate producing plants older or matureplant tissue may also be used. The obtained cDNA libraries can be probedwith antibodies specifically binding the cytochrome P-450 monooxygenaseand clones expressing the monooxygenase can be isolated.

An alternative method for the preparation of cDNA coding for acytochrome P-450 monooxygenase comprises

(a) isolating and solubilizing microsomes from plant tissue producingcyanogenic glycosides or glucosinolates,

(b) purifying the cytochrome P-450 monooxygenase,

(c) obtaining a complete or partial protein sequence of themonoxygenase,

(d) designing oligonucleotides specifying DNA coding for 4 to 15 aminoacids of said monooxygenase protein sequence

(e) probing a cDNA library of plant tissue producing cyanogenicglycosides or glucosinolates with said oligonucleotides, or DNAmolecules obtained from PCR amplification of cDNA using saidoligonucleotides, and

(f) isolating clones which encode cytochrome P-450 monooxygenase.

Amino acid sequences of internal peptides which are the result ofprotease digestion can be obtained by standard techniques such as Edmandegradation. Oligonucleotides specifying DNA coding for partial proteinsequences of the inventive monooxygenases are obtained by reversetranslation of parts of the protein sequence according to the geneticcode. Protein sequences encoded by DNA sequences of low degeneracy arepreferred for reverse translation. Their length ranges from 4 to 15 andpreferably from 5 to 10 amino acids. If necessary the codons used in theoligonucleotides can be adapted to the codon usage of the plant source(Murray et al, Nucleic Acids Research 17:477-498, 1989). The obtainedoligonucleotides can be used to probe cDNA libraries as described inSambrook et al, Molecular cloning: A laboratory manual. Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., 1989, for cloneswhich are able to basepair with said oligonucleotides. Alternatively,oligonucleotides can be used in a polymerase chain reaction, themethodology of which is known in the art, with plant cDNA as thetemplate for amplification. In this case the obtained amplificationproducts are used to probe the cDNA libraries. Clones encodingcytochrome P-450 monooxygenases are isolated.

An alternative method of cloning genes is based on the construction of agene library composed of expression vectors. In that method, analogouslyto the methods already described above, genomic DNA, but preferablycDNA, is first isolated from a cell or a tissue capable of expressing adesired gene product--in the present case a P-450 monooxygenase--and isthen spliced into a suitable expression vector. The gene libraries soproduced can then be screened using suitable measures, preferably usingantibodies, and those clones selected which comprise the desired gene orat least part of that gene as an insert.

Alternatively, total DNA from the DNA library, preferably from the cDNAlibrary, can be prepared and used as a template for a PCR reaction withprimers representing low degeneracy portions of the amino acid sequence.Preferably, the primers used will generate PCR products that represent asignificant portion of the nucleotide sequence. The PCR products can befurther probed to determine if they correspond to a portion of the P-450monooxygenase gene using a synthetic oligonucleotide probe correspondingto an amino acid fragment sequence located in the interior or middleregion of the P-450 monooxygenase protein.

The cDNA clone s an d PCR products prepared as described above orfragments thereof may be used as a hybridization probe in a process ofidentifying further DNA sequences from a homologous or a heterologoussource organism encoding a protein product that exhibits P-450monooxygenase activity such as, for example, a fungi or a heterologousplant. A suitable source would be tissue from plants containingcyanogenic glycosides or glucosinolates.

They may also be used as an RFLP marker to determine, for example, thelocation of the cytochrome P-450 monooxygenase gene or a closely linedtrait in the plant genome or for marker assisted breeding EP-A 306,139;WO 89/07647!.

Using the methods described above it is thus possible to isolate a genethat codes for a P-450 monooxygenase.

Genes encoding cytochrome P-450 monooxygenase can be used in a methodfor producing a purified recombinant cytochrome P-450 monooxygenasewhich monooxygenase either catalyzes the conversion of an amino acidpreferably selected from the group consisting of tyrosine,phenylalanine, tryptophan, valine, leucine, isoleucine andcyclopentenylglycine to the corresponding N-hydroxyamino acid and theoxime derived from this N-hydroxyamino acid; or the conversion of saidoxime to a nitrile and the conversion of said nitrile to thecorresponding cyanohydrine; comprising

(a) engineering the gene encoding said monooxygenase to be expressiblein a host organism such as bacteria, yeast or insect cells,

(b) transforming said host organism with the engineered gene, and

(c) isolating the protein from the host organism or the culturesupernatant.

In a preferred embodiment of the invention the method is used to obtainpurified recombinant cytochrome P-450_(TYR), P-450_(Ox) or cytochromeP-450_(TYR) which has been modified by known techniques of genetechnology. Preferably the modifications lead to increased expression ofthe recombinant protein or to altered substrate specificity.

The inventive DNA molecules can be used to obtain transgenic plantsresistant to insects or acarids examples of which are listed but notlimited to those in Table B as well as nematodes. Preferably thetransgenic plants are resistant to Coleoptera and Lepidoptera such aswestern corn root worm (Diabrotica virgifera virgifera), northern cornroot worm (Diabrotica longicornis barberi), southern corn rootworm(Diabrotica undecimpunctata howardi), cotton bollworm, European cornborer, corn root webworm, pink bollworm and tobacco budworm. Thetransgenic plants comprise DNA coding for monooxygenases which catalyzethe conversion of an amino acid to the corresponding N-hydroxyamino acidand the oxime derived from this N-hydroxyamino acid; or the conversionof said oxime to a nitrile and the conversion of said nitrile to thecorresponding cyanohydrine. In addition the transgenic plants maycomprise monooxygenase genes genetically linked to herbicide resistancegenes. The transgenic plants are preferably monocotyledoneous ordicotyledoneous plants examples of which ar listed in Table A.Preferably they are selected from the group consisting of maize, rice,wheat, barley, sorghum, cotton, soybeans, sunflower, grasses and oilseed rape. The plants can be obtained by a method comprising

(a) introducing into a plant cell or plant tissue which can beregenerated to a complete plant, DNA comprising a gene expressible inthat plant encoding an inventive monooxygenase,

(b) selecting transgenic plants, and

(c) identifying plants which are resistant to insects, acarids, ornematodes.

The inventive DNA molecules can also be used to obtain transgenic plantsexpressing anti-sense or sense RNA or ribozymes targeted to the genes ofthe endogenous P-450 monooxygenases the expression of which reduces theexpression of cytochrome P-450 monooxygenases. Such plants show improveddisease resistance or nutritive value due to reduced expression ofcyanogenic glycosides or glucosinolates. The plants can be obtained witha method comprising

(a) introducing into a plant cell or tissue which can be regenerated toa complete plant, DNA encoding sense RNA, anti sense RNA or a ribozyme,the expression of which reduces the expression of cytochrome P-450monooxygenases according to claims 1 or 8,

(b) selecting transgenic plants, and

(c) identifying plants with improved disease resistance or nutritivevalue.

A number of very efficient processes are available for introducing DNAinto plant cells, which processes are based on the use of gene transfervectors or on direct gene transfer processes.

One possible method of inserting a gene construct into a cell makes useof the infection of the plant cell with Agrobacterium tumefaciens and/orAgrobacterium rhizogenes, which has been transformed with the said geneconstruction. The transgenic plant cells are then cultured undersuitable culture conditions known to the person skilled in the art, sothat they form shoots and roots and whole plants are finally formed.

Within the scope of this invention is the so-called leaf disktransformation using Agrobacterium (Horsch et al, Science 227:1229-1231,1985) can also be used. Sterile leaf disks from a suitable target plantare incubated with Agrobacterium cells comprising one of the chimaericgene constructions according to the invention, and are then transferredinto or onto a suitable nutrient medium. Especially suitable, andtherefore preferred within the scope of this invention, are LS mediathat have been solidified by the addition of agar and enriched with oneor more of the plant growth regulators customarily used, especiallythose selected from the group of the auxins consisting ofa-naphthylacetic acid, picloram, 2,4,5-trichlorophenoxyacetic acid,2,4-dichlorophenoxyacetic acid, indole-3-butyric acid, indole-3-lacticacid, indole-3-succinic acid, indole-3-acetic acid andp-chlorophenoxyacetic acid, and from the group of the cytokininsconsisting of kinetin, 6-benzyladenine, 2-isopentenyladenine and zeatin.The preferred concentration of auxins and cytokinins is in the range of0.1 mg/l to 10 mg/l.

After incubation for several days, but preferably after incubation for 2to 3 days at a temperature of 20° C. to 40° C., preferably from 23° C.to 35° C. and more preferably at 25° C. and in diffuse light, the leafdisks are transferred to a suitable medium for the purpose of shootinduction. Especially preferred for the selection of the transformantsis an LS medium that does not contain auxin but contains cytokinininstead, and to which a selective substance has been added. The culturesare kept in the light and are transferred to fresh medium at suitableintervals, but preferably at intervals of one week. Developing greenshoots are cut out and cultured further in a medium that induces theshoots to form roots. Especially preferred within the scope of thisinvention is an LS medium that does not contain auxin or cytokinin butto which a selective substance has been added for the selection of thetransformants.

In addition to Agrobacterium-mediated transformation, within the scopeof this invention it is possible to use direct transformation methodsfor the insertion of the gene constructions according to the inventioninto plant material.

For example, the genetic material contained in a vector can be inserteddirectly into a plant cell, for example using purely physicalprocedures, for example by microinjection using finely drawnmicropipettes (Neuhaus et al, Theoretical and Applied Genetics74:363-373, 1987), electroporation (D'Halluin et al, The Plant Cell4:1495-1505, 1992; WO 92/09696), or preferably by bombarding the cellswith microprojectiles that are coated with the transforming DNA("Microprojectile Bombardment"; Wang et al, Plant Molecular Biology11:433-439, 1988; Gordon-Kamm et al, The Plant Cell 2:603-618, 1990;McCabe et al, Bio/Technology 11:596-598, 1993; Christou et, PlantPhysiol. 87:671-674, 1988; Koziel et al, Biotechnology 11: 194-200,1993). Moreover, the plant material to be transformed can optionally bepretreated with an osmotically active substance such as sucrose,sorbitol, polyethylene glycol, glucose or mannitol.

Other possible methods for the direct transfer of genetic material intoa plant cell comprise the treatment of protoplasts using procedures thatmodify the plasma membrane, for example polyethylene glycol treatment,heat shock treatment or electroporation, or a combination of thoseprocedures (Shillito et al, Biotechnology 3:1099-1103, 1985).

A further method for the direct introduction of genetic material intoplant cells, which is based on purely chemical procedures and whichenables the transformation to be carried out very efficiently andrapidly, is described in Negrutiu et al, Plant Molecular Biology8:363-373, 1987.

Also suitable for the transformation of plant material is direct genetransfer using co-transformation (Schocher et al, Bio/Technology4:1093-1096, 1986).

The list of possible transformation methods given above by way ofexample does not claim to be complete and is not intended to limit thesubject of the invention in any way.

In another embodiment of the invention target plants are exposed to apesticidally effective amount of a cyanogenic glycoside to controlinsects, acarids, or nematodes attacking a monocotyledonous ordicotyledonous plant selected from the group of plant types consistingof Cereals, Protein Crops, Fruit Crops, Vegetables and Tubers, Nuts, OilCrops, Sugar Crops, Forage and Turf Grasses, Forage Legumes, FiberPlants and Woody Plants, Drug Crops and Spices and Flavorings.

The following examples further describe the materials and methods usedin carrying out the invention and the subsequent results. They areoffered by way of illustration, and their recitation should not beconsidered as a limitation of the claimed invention.

EXAMPLES Example 1

Preparation of microsomes

Seeds of Sorghum bicolor (L.) Moench (hybrid S-1000) are obtained fromSeedtec International Inc. (Hereford, Tex.) and germinated in the darkfor 2 days at 28° C. on metal screens covered with gauze. Transfer ofthe seeds to germination trays is carried out under dim green light.Microsomes are prepared from approximately 3 cm tall etiolatedseedlings. The seedlings are harvested and homogenized using a mortarand pestle in 2 volumes (v/w) of 250 mM sucrose, 100 mM tricine (pH7,9), 50 mM NaCl, 2 mM EDTA and 2 mM DTT. Polyvinylpolypyrrolidone isadded (0.1 g/g fresh weight) prior to homogenization. The homogenate isfiltered through 22 μm nylon cloth and centrifuged 20 minutes at 48000g. The supernatant is centrifuged for 1 hour at 165000 g. The microsomalpellet is resuspended and homogenized in isolation buffer using aPotter-Elvehjem homogenizer fitted with a teflon pestle. Afterrecentrifugation and rehomogenization, the homogenate is dialyzedovernight against 50 mM Tricine (pH 7,9), 2 mM DTT under a nitrogenatmosphere.

Example 2

Enzyme assays: Determination of total cytochrome P-450

Quantitative determination of total cytochrome P-450 is carried out bydifference spectroscopy using an extinction difference coefficient of 91mM⁻¹ cm⁻¹ for the complex between reduced cytochrome P-450 and carbonmonoxide(A₄₅₀₋₄₉₀) (Omura et al, J. Biol. Chem. 239:2370-2378, 1964).Cytochrome P-450 substrate binding spectra are recorded with stepwiseincreased substrate concentration until saturating conditions arereached.

Example 3

Purification of cytochrome P-450_(TYR) and P-450_(Ox)

    ______________________________________    Buffer A   Buffer B     Buffer C    ______________________________________    8.6% glycerol               8.6% glycerol                            8.6% glycerol    10 mM      40 mM        40 mM KH.sub.2 PO.sub.4 /K.sub.2 HPO.sub.4    KH.sub.2 PO.sub.4 /K.sub.2 HPO.sub.4               KH.sub.2 PO.sub.4 /K.sub.2 HPO.sub.4                            (pH 7.9)    (pH 7.9)   (pH 7.9)    0.20 mM EDTA               5.0 mM EDTA  5.0 mM EDTA    2.0 mM DTT 2.0 mM DTF   2.0 mM DTT    1.0% RENEX 690               1.0% RENEX 690                            1.0% CHAPS    0.05% RTX-100               0.05% RTX-100                            0.05% RTX-100               0.2% CHAPS    ______________________________________

Buffers are degassed three times by stirring in vacuo before detergentand DTT are added. Between each degassing, the buffer is flushed withargon.

Microsomes (400 mg protein in 20 ml) are diluted to 100 ml with a buffercomposed of 8.6% glycerol, 10 mM KH₂ PO₄ /K₂ HPO₄ (pH 7.9). Themicrosomes are solubilized by slow addition of 100 ml of the same buffercontaining 2% RENEX 690 and 0.2% RTX-100 and constant stirring for 30minutes. Solubilized cytochrome P-450 is obtained as the supernatantafter centrifugation for 30 minutes at 200 000 g in a Beckman 70:Tirotor. The supernatant (190 ml) is applied (flow rate 100 ml/h) to acolumn (5×5 cm) of DEAE Sepharose fast flow/S-100 Sepharose (20:80 wetvolumes) equilibrated in buffer A. The ion exchange resin DEAE-Sepharoseis diluted with the gel filtration material Sephacryl S-100 in the ratio1:4 to avoid too high concentrations of the cytochrome P-450 enzymesupon binding, which sometimes results in irreversible aggregation. Thecolumn is washed with 150 ml buffer A after which the total amount ofcytochromes P-450 including cytochrome P-450_(TYR) and cytochromeP-450_(Ox) is eluted with buffer B in a total volume of 150 ml. Duringthis procedure, NADPH-cytochrome P-450-oxidoreductase and Cytochrome b₅remain bound to the column and may subsequently be eluted and separatedwith buffer B and a gradient of 0-300 mM KCl.

The cytochrome P-450 eluate is adjusted to 1.0% CHAPS, stirred for 30minutes and then directly applied to a 25 ml (2.6×5 cm) column ofReactive yellow 3 sepharose equilibrated in buffer C+1,0% RENEX 690. Theflow rate used is 25 ml/h. The column is washed with buffer C until theabsorbance A₂₈₀ shows that RENEX 690 is washed out. CytochromeP-450_(TYR) does not bind to this column, and is obtained in the run-offand wash. Subsequently the column is eluted with 400 mM KCl in buffer C.The cytochrome P-450_(Ox) containing fractions are combined yieldingapproximately 60 ml and diluted with 5 volumes of buffer C to lower theKCl strength and permit rebinding of cytochrome P-450_(Ox) on a secondReactive yellow 3 column. This column is eluted with a KCl gradient(0-500 mM) in a total volume of 100 ml in buffer C. This serves to elutecytochrome P-450_(Ox).

The cytochrome P-450_(Ox) pool from the yellow agarose is diluted 5times with buffer C to 20-25 mM KCl and applied to a Cibachron blueagarose column (0.9×6 cm) equilibrated in buffer C. The flow rate usedis 8 ml/h. The column is washed with 20 ml buffer C at the same flowrate. Cytochrome P-450_(Ox) is eluted with a gradient of KCl, 0-2.0M inbuffer C in a total volume of 30 ml.

The runoff from the first yellow 3 agarose column is applied (flow rate40 ml/h) to a column (2.8×8 cm) of Cibachron Blue Agarose equilibratedin buffer C. The column is subsequently washed with buffer C and thecytochrome P-450_(TYR) is eluted with a 0-500 mM linear KCl-gradient(2×100 ml) in buffer C. The combined cytochrome P-450 fractions arediluted 5 times with buffer C and applied (flow rate 7 ml/h) to a column(0.9×5 cm) of Reactive red 120 agarose equilibrated in buffer C. Thecolumn is washed with 25 ml buffer C and cytochrome P-450_(TYR) iseluted with a 0-1.0M KCl linear gradient (2×30 ml) in buffer C.Optionally the eluate is gelfiltrated through a Sephadex G-50 column,equilibrated in a buffer composed of 50 mM potassium phosphate (pH7.9)/400 mM KCl/0.1% CHAPS/2 mM DTT. The eluted cytochrome P-450_(TYR)is dialyzed for 2 hours against 50 mM potassium phosphate (pH 7.9)/2 mMDTT, diluted 4 fold with dialysis buffer in an Amicon ultrafiltrationcell fitted with a YM-30 membrane and concentrated to 1.45 nmols/ml.

All procedures are carried out at 4° C. The total cytochrome P-450content of the individual fractions is determined from the carbonmonoxide difference spectrum. The absorption spectrum of the oxidizedcytochrome P-450 is also recorded. The presence of a specific cytochromeP-450 is monitored by substrate binding spectra

Example 4

Antibody preparation

Polyclonal antibodies are elicited in rabbits by six repeatedsubcutaneous injections (approx. 15 μg protein per rabbit per injection)at 15 day intervals of cytochrome P-450_(TYR) or P-450_(Ox) isolated bydye column chromatography or denatured enzyme purified by preparativeSDS-PAGE. Freunds complete adjuvant is included in the first injectionwhereas Freunds incomplete adjuvant is used in subsequent injections.The immunoglobulin fractions of the antisera are purified by ammoniumsulfate precipitation (Harboe et al, `A Manual of QuantitativeImmunoelectrophoresis: Methods and Applications`, Universitetsforlaget,Oslo, 1973). The antiobides are monospecific as demonstrated by Westernblotting.

Example 5

Characterization of P-450_(TYR)

5.1. Substrate binding spectra of P-450_(TYR)

Cytochrome P-450_(TYR) in the oxidized state has a strong absorptionpeak at 420 nm, representing the low spin state of the iron of the hemegroup. The binding of a ligand to the heme group shifts the absorptionmaximum by changing the spin state of the iron. Binding of tyrosine atthe catalytic site of cytochrome P-450_(TYR) induces a change of thespin state of the oxidized iron from low to high spin, and therebychanges the absorption maximum from 420 nm to 390 nm producing a type Ispectrum (Jefcoate, Methods in Enzymology 52:258-279, 1978). Thefollowing experimental procedure is used to obtain the substrate bindingspectrum: two identical cuvettes containing a buffered solution of theisolated cytochrome P-450 are prepared. The substrate of the enzyme isadded to the sample cuvette whereas the same volume of buffer is addedto the other cuvette. The difference spectrum is then recorded in anSLM-Aminco DW2c spectrophotometer. The absorption difference, A390-420,is proportional to the concentration of cytochrome P-450_(TYR) with abound substrate at its active site. If a saturating concentration ofsubstrate is added to the sample cuvette, the absorption difference isproportional to the concentration of the substrate specific cytochromeP-450 in the cuvettes. The saturating concentration of the substrate isdetermined by titrating the cytochrome P-450 sample with increasingamounts of substrate and monitoring A₃₉₀₋₄₂₀.

If a cytochrome P-450 sample can be saturated with two differentsubstrates, there may be two different cytochrome P-450 enzymes in thesample, or there may be one cytochrome P-450 enzyme able to bind to bothsubstrates. To discriminate between these possibilities, saturatingamounts of the two substrates are added sequentially and the A₃₉₀₋₄₂₀absorption change is monitored. If, independent of the order ofaddition, the addition of the second sample gives rise to an increasedA₃₉₀₋₄₂₀ value compared to the value after the addition of the firstsubstrate, the two substrates are bound by different enzymes. IfA₃₉₀₋₄₂₀ remains unchanged upon addition of the second substrate,independent of the order of addition, both substrates bind to the sameactive site, i.e. to the same cytochrome P-450 enzyme. The data shown inTables C and D below represent results of a typical experiment.

                  TABLE C    ______________________________________    To 500 μl of isolated cytochrome P-450.sub.TYR dissolved in 50 mM    Tricine pH 7,9 tyrosine is added until saturation concentration is    reached    followed by addition of N-hydroxytyrosine:                  initial               resulting    Added substrate                  A.sub.390-420                           dilution factor                                        A.sub.390-420    ______________________________________    30 μl 5 mM tyrosine                  0,0437   530/500      0,0463    60 μl 5 mM tyrosine                  0,0496   560/500      0,0556    90 μl 5 mM tyrosine                  0,0486   590/500      0,0573    + 100 μl 20 mM                  0,0409   690/500      0,0564    N-hydroxytyrosine    ______________________________________

                  TABLE D    ______________________________________    Addtion of N-hydroxytyrosine until saturation concentration is reached,    followed by addition of tyrosine                 initial               resulting    Added substrate                 A.sub.390-420                           dilution factor                                       A.sub.390-420    ______________________________________    50 μl 20 mM                 0,0689    550/500     0,0758    N-hydroxytyrosine    120 μl 5 mM                 0,0919    620/500     0,1140    N-hydroxytyrosine    140 μl 5 mM                 0,0911    640/500     0,1166    N-hydroxytyrosine    + 90 μl 5 mM tyrosine                 0,0726    730/500     0,1060    ______________________________________

Both tyrosine and N-hydroxytyrosine produce a type I binding spectrum.The data show, that tyrosine and N-hydroxytyrosine bind to the sameactive site, that is the same cytochrome P-450, thus demonstrating thatcytochrome P-450_(TYR) is multifunctional. From the amounts ofcytochrome P-450_(TYR) used the absorption coefficient (ε₃₉₀₋₄₂₀) iscalculated to be 67 cm⁻¹ mM⁻¹. A complete transition from a low spinstate to a high spin state would have resulted in an absorptioncoefficient of 138 cm⁻¹ mM⁻¹.

5.2. Molecular weight and Amino acid sequence data

The molecular weight of P-450_(TYR) as determined by SDS-PAGE is 57 kD.

Amino acid sequences are obtained by automated Edman degradation. Theinternal polypeptides are obtained by trypsin digestion of the purifiedprotein and subsequent separation of the peptides using reverse phaseHPLC.

N-terminal sequence:

    --MATMEVEAAAATVLAAP--                                      (SEQ ID NO:3)

Internal sequences:

    --VWDEPLR--                                                (SEQ ID NO: 4)

    --YVYNLATK--                                               (SEQ ID NO: 5)

    --SDTFMATPLVSSAEPR--                                       (SEQ ID NO: 6)

    --AQSQDITFAAVDNPSNAVEXALAEMVNNPEVMAK--                     (SEQ ID NO: 7)

    --AQGNPLLTIEEVK--                                          (SEQ ID NO: 8)

    --LVQESDIPK--                                              (SEQ ID NO: 9)

    --ISFSTG--                                                 (SEQ ID NO: 10)

    --LPAHLYPSISLH--                                           (SEQ ID NO: 11)

5.3. Reconstitution of cytochrome P-450_(TYR) activity:

Reconstitution of the enzyme activity of a microsomal P-450 enzyme isaccomplished by insertion of the cytochrome P-450 enzyme and thecorresponding NADPH-cytochrome P-450 oxidoreductase into appropriatelipid micelles made from different commercially available lipids.Isolation of NADPH-cytochrome P-450 oxidoreductase is done according toHalkier and Moller, Plant Physiol. 96:10-17, 1990. A mixture of lipidscan be used but with cytochrome P-450_(TYR) di-lauroyl-phosphatidylcholine (DLPC) provides the best enzymatic activity. One rate limitingfactor of this rate limiting reaction is the number of correctly formedcomplexes of cytochrome P-450_(TYR) and NADPH-cytochrome P-450oxidoreductase. Excess amounts of the oxidoreductase and concentratedenzyme solutions ensure a sufficient number of active complexes.

A reconstituted enzyme is obtained using the following components:

Cytochrome P-450_(TYR) : 100 μg/ml in 50 mM potassium phosphate bufferpH 7,9

Oxidoreductase, purified from Sorghum bicolor: 100 μg/ml in 50 mMpotassium phosphate buffer pH 7,9

Lipid: 10 mg/ml di-lauroyl-phosphatidyl choline, sonicated in 50 mMpotassium phosphate buffer pH 7,9

NADPH: 25 mg/ml H₂ O

¹⁴ C-tyrosine: commercially available from Amersham; U-¹⁴ C!-L-tyrosine0.5 μCi

10 μl of the lipid suspension is mixed in a glass vial with 50 μl of thecytochrome P-450_(TYR) (0-1.5 pmol) solution. 50 μl of theoxidoreductase (0-0.15 U) solution is added and then 10 μl tyrosinesolution and 10 μl NADPH solution are added and the mixture is sonicatedin a Branson 5200 sonication bath for one minute. The reaction mixtureis subsequently incubated for 1 hour at 30° C. At the end of theincubation period the reaction is stopped by transferring the glassvials onto ice. Radioactively labelled intermediates formed areextracted into 50 μl ehtyl acetate, applied to a silica coated TLC plateand developed using an ethyl acetate/toluene (1:5 v/v) mixture as mobilephase. The resultant product, p-hydroxyphenylacetaldehyde oxime isvisualized by autoradiography of the TLC plate. Alternatively, theintermediates are analyzed by reverse-phase HPLC coupled to a Bertholdradioactiviy monitor. The HPLC separation was carried out using anucleosil 100-10C₁₈ column isocratically eluted with 1,5% 2-propanol in25 mM Hepes pH 7,9 (Halkier et al, J. Biol. Chem. 264:19487-19494,1989). Control samples may be made by omitting either cytochrome P-450oxidoreductase or NADPH.

When reconstituted into micelles cytochrome P-450_(TYR) catalyzes theconversion of L-tyrosine all the way to p-hydroxyphenyl-acetaldehydeoxime. The K_(m) and turn-over number of the enzyme are 0.14 mM and 198min⁻¹, respectively, when assayed in the presence of 15 mM NaCl, whereasthe values are 0.21 mM and 228 min⁻¹ when assayed in the absence ofadded salt.

The formation of p-hydroxyphenyl-acetaldehyde oxime demonstrates thatcytochrome P-450_(TYR) is a multi-functional heme-thiolate proteincatalyzing reactions in addition to the initial N-hydroxylation ofL-tyrosine. The E/Z ration of the parahydroxyphenyl-acetaldehyde oximeproduced by the reconstituted cytochrome P-450_(TYR) and determined byHPLC chromatography is 69/31. Using the TLC/autoradiography system,minute amounts of radiolabelled products comigrating with authenticp-hydroxybenzaldehyde and 1-nitro-2(p-hydroxyphenyl)ethane are detectedin the reaction mixtures.

5.4. Inhibitory effect of antibodies against cytochrome P-450_(TYR)

The experiments are carried out using monospecific antibodies againstP-450_(TYR) as described in section 6.4. which uses antibodies againstcytochrome P-450_(Ox). The results are similiar to those obtained withthe antibody against cytochrome P-450_(Ox) except that the cytochromeP-450_(TYR) antibody exerts a stronger inhibitory effect (up to 60%) oncyanide production.

5.5. cDNA libraries and colony screening

Poly A⁺ RNA is isolated from 3 cm high etiolated seedlings of Sorghumbicolor (L.) Moench grown as described for seedlings used forpreparation of microsomes. The poly A⁺ RNA is used for the constructionof a λgt11 expression library and a λgt10 library. The construction ofthe libraries can be done according to the procedures described forexample in Sambrook et al, Molecular cloning: A laboratory manual. ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989 or can beordered by commercial companies such as Strategene (La Jolla, Calif.).Antibodies obtained against cytochrome P450_(TYR) are used to screen theexpression libraries as described by Young et al, Procl. Natl. Acad. SciUSA 82:2583-2587. Antigen-antibody complexes are detected enzymaticallywith alkaline phosphatase-conjugated antibodies (Dakopatts). DNA from 4positive plaques is prepared according to Grossberger, Nucleic AcidResearch 15:6737, 1987. Inserts from λ phages are subcloned intopBluescript II SK (Strategene). Comparison of the deduced amino acidsequence from one of the four inserts with the amino acid sequencesobtained from protein sequencing of P450_(TYR) shows that this clone isa partial cDNA clone for P450_(TYR). The partial cDNA clone is used as aprobe for a new screen of the λgt10 and λgt11 libraries. The insertsizes of 45 positive clones are determined by southern blotting.Additionally the 45 positive clones are examined for hybridization withtwo different mixtures of oligonucleotides by southern blotting. Thesequences of the oligonucleotide mixtures are based on the partial aminoacid sequence data and specify a sequence near the N-terminal end (aminoacids 4 to 9) and a sequence near the C-terminal end (amino acids533-538). Oligonucleotide synthesis is carried out on a Cyclone Plus DNASynthesizer. Sequencing of one clone derived from the λgt10 libraryshowing the expected size and hybridizing with the two oligonucleotidemixtures shows that the clone is a full-length cDNA clone encodingcytochrome P450_(TYR).

Oligonucleotide specifying amino acids 4 to 9 (MEVEAA) (SEQ ID NO: 17)

5'-ATG-GA G,A!-GT C,G,T,A!-GA G,A!-GC CGTA!-GC-3'

Oligonucleotide specifying amino acids 533 to 538 (DFTMAT) (SEQ ID NO:18)

5'-GA C,T!-AC C,G,T,A!-TT C,T!-ATG-GC C,G,T,A!-AC-3'

5.6. DNA sequencing

DNA sequencing is carried out by the dideoxy chain method (Sanger et al,Proc. Natl. Acad. Sci. USA 74:5463-5467, 1977) using ³⁵ S!-dATP. T7 DNApolymerase and deoxynucleotides are obtained from Pharmacia,dideoxynucleotides from Boehringer Mannheim. Sequencing of thefull-length cDNA clone is done partly by subcloning and partly by usingsynthetic oligonuecleotides as primers. The oligonucleotide primers canbe ordered with commerical companies.

5.7. Southern blotting

λDNA isolated from the positive clones is digested with Eco RI. Theinserts are separated from λDNA by electrophoresis on a 0.7% agarosegel. After electrophoresis, DNA is capillary blotted onto a Zetaprobemembrane (Biorad) using 10 mM NaOH for the transfer. Hybridization isperformed at 68° C. in 1.5×SSPE (270 mM NaCl, 15 mM Na₂ HPO₄ pH 7.0, 1.5mM EDTA, 1% sodium dodecyl sulphate) 10% dextransulphate, 0,5% skim milkand 0.1 mg/ml salmon sperm DNA for 16 hours. When the partial cDNA cloneis used as probe for hybridization it is labeled with α-³² P!dCTP usinga random prime labelling kit (Amersham International plc.). Theoligonucleotide mixtures are 5'end labeled according to Okkels et al.(Okkels et al, FEBS Letters 237:108-112, 1988). The filters are washedfirst in 2×SSC (0.9M NaCl, 0.09M trisodium citrate, 0.1% SDS) at 47° C.for 15 min., then in a fresh solution of the same composition at 56° C.for 15 min. and finally in 0.1×SSPE, 0.5% SDS for 30 minutes at 65° C.The presence of radioactively labelled hybridization bands on the filteris monitored by X-ray autoradiography.

5.8. Characterization of a full-length cDNA clone

λDNA isolated from the positive clones is digested with Eco RI. Theinsert is separated from λDNA by electrophoresis on a 0.7% agarose geland subcloned into the EcoRI site of the vector pBluescript SK(Strategene) contained within the sequence GCAGGAATTCCGG (SEQ ID NO:24). The four last bases of this sequence are listed as the first fourbases in SEQ ID NO: 1. A clone comprising the described cDNA has beendeposited with the Agricultural Research Culture Collection (NRRL), 1815N. University Street, Peoria, Ill. 61604 U.S.A. under the accessionnumber NRRL B-21168.

The orientation of the insert in the vector is determined as thepolylinker restriction site for Pst I being adjacent to the 5'end of thecytochrome P-450 sequence. The sequence of the insert is shown in SEQ IDNO: 1. The sequence comprises an open reading frame (ORF) starting atnucleotide 188 and ending at nucleotide 1861 of SEQ ID NO: 1. It encodesa protein of 558 amino acids and a molecular mass of 61887 Da shown inSEQ ID NO: 2. The sequence comprises the sequences of SEQ ID NO: 3 toSEQ ID NO: 11. The protein is not subject to post-translationalmodification at the N- and C-terminal ends except for the removal of theN-terminal methionine residue. The N-terminal region of cytochromeP-450_(TYR), however, shows four motifs which in animals are known totarget heme-thiolate proteins to the endoplasmatic reticulum.

Searches for sequence similarity are made using the programmes BLAST andFASTA in the nucleotide sequence data bases provided by the EMBL.Pairwise comparisons of cytochrome P-450_(TYR) with other cytochromeP-450 sequences are performed using the programme GAP of the GeneticsComputer Group GCG sofware package. Multiple alignments were made usingthe GCG programme PILEUP.

5.9. Expression of native cytochrome P-450_(TYR) in E. coli

Plasmid pCWOri+ (Gegner et al, Prod. Natl. Acad. Sci. USA 88:750-754,1991) is used to express the wildtype cytochrome P-450_(TYR) cDNAsequence as described by Barnes et al, Prod. Natl. Acad. Sci. USA80:5597-5601, 1991. cDNA sequences are introduced into the expressionplasmid using polymerase chain reaction (PCR) mutagenesis. A syntheticoligonucleotide (TYROL1b) containing an amino acid-conserving andnucleotide modifying 5' cDNA sequence is used in conjunction with adownstream oligonucleotide (TYROL3) to amplify the N-terminal sequencebetween the ATG initiator codon (contained within an NdeI site) and aunique BamHI restriction site within the cytochrome P-450_(TYR)sequence. A synthetic oligonucleotide (TYROL2) is used in conjunctionwith an oligonucleotide (TYROL4) complementary to a unique PstIrestriction site to introduce a HindIII restriction site immediatelydownstream of the TGA stop codon. The expression plasmid pCWtyr isconstructed by simultaneous ligation of the 278 basepair PCR NdeI/BamHIfragment, the 1257 basepair BamHI/PstI fragment of cytochromeP-450_(TYR) and the 146 basepair PCR PstI/HindIII fragment with theNdeI/HindIII cleaved vector DNA. E. coli strain JM 109 transformed withplasmid pCWtyr is grown in LB/ampicilline medium at 37° C. Expression ofcytochrome P-450_(TYR) is obtained by growing the cells in a mediumcontaining 1 mM isopropyl beta-D-thiogalactopyranoside (IPTG) andshifting the cells to growth at 28° C. at 125 rpm. E. coli produces afunctionally active cytochrome P-450_(TYR) enzyme which convertstyrosine into oxime. The analytical procedures are as in thereconstitution experiments described in section 5.3. above. Theexpressed cDNA clone encoding P-450_(TYR) specifies the synthesis of asingle cytochrome P-450 enzyme. Since this enzyme catalyzes theconversion of tyrosine all the way to p-hydroxyphenylacetaldehyde oxime,this unambigeously demonstrates that cytochrome P-450_(TYR) ismultifunctional.

The following oligonucleotides are used:

TYROL1b (SEQ ID NO: 19)

5'-CGG GAT CCA TAT GCT GCT GTT ATT AGC AGT TTT TCT GTC GTA-3'

TYROL2 (SEQ ID NO: 20)

5'-GAC CGG CCG AAG CCT TAA TTA GAT GGA GAT GGA-3'

TYROL3 (SEQ ID NO: 21)

5'-AGT GGA TCC AGC GGA ATG CCG GCT T-3'

TYROL4 (SEQ ID NO: 22)

5'-CGT CAT GCT CTT CGG AA-3'

5.10. Expression of truncated and modified cytochrome P-450tyr in E.coli

A modified cytocrome P-450_(TYR), in which the 35 N-terminal amino acidsare replaced by the nine N-terminal amino acids from bovine 17αhydroxylase is introduced into the expression vector pSP19g10L which canbe obtained from Dr. Henry Barnes (La Jolla, Calif.). This plasmidcontains the lac Z promoter fused with the known short leader sequence(g10L) of gene 10 from bacteriophage T₇ (Olin et al, 1988). A constructcontaining the N-terminal amino acids from bovine 17α hydroxylase and atruncated form of the P-450_(TYR) gene is designed using PCRmutagenesis: Oligonucleotide TYROL 1d (5'-CGG GAT CCA TAT GGC TCT GTTATT AGC AGT TTT TCT GTC GTA CCT GGC CCG-3'; SEQ ID NO 23) containing a5' mutant cDNA sequence as well as a BamHI and NdeI restriction site isused together with oligonucleotide TYROL 3 comprising the sequencesurrounding the unique BamHI restriction site downstream of the ATGstart codon of the cDNA coding for P-450_(TYR) to amplify a modifiedN-terminal sequence of P-450_(TYR). The amplification product is cutwith NdeI and BamHI restriction enzymes. To introduce a HindIII siteimmediately downstream of the stop codon of the P-450_(TYR) gene,oligonucleotides TYROL 2 and TYROL 4 are used in a polymerase chainreaction to obtain a C-terminal fragment of the P-450_(TYR) genecomprising a HindIII restriction site immediately downstream of the stopcodon. The amplification product is cut with PstI and HindIIIrestriction enzymes. The complete expression plasmid is constructed bysimultaneous ligation of the N-terminal NdeI/BamHI fragment, theBamHI/PstI fragment of the P-450_(TYR) gene and the C-terminalPstI/HindIII fragment into NdeI/HindIII cleaved pSP19g10L vector DNA.The expression vector obtained is transformed into E. coli strain JM109.Transformed E. coli produce 300 nmol to 500 nmol cytochrome P-450_(TYR)per liter cell culture upon growth at 28° C. in the presence of 1 mMisopropyl-β-D-thiogalactopyranoside and at 125 rpm. Expression levels ashigh as 900 nmol per liter, equivalent to 55 mg P-450_(TYR) per liter,have been obtained.

Administration of tyrosine to the cell culture results in the productionof p-hydroxyphenylacetaldehyde oxime, whereas a cell culture transformedwith pSP19g10L alone does not produce the oxime.

Reconstitution experiments with E. cole-expressed cytochrome P-450_(TYR)and sorghum NADPH cytochorme P450 reductase indilaurylpohsphatidylcholine micelles is performed as described insection 5.3. above. Turnover rates of 349 nmol oxime per nmolP-450_(TYR) per minute can be demonstrated, which is equivalent to thevalues obtained with sorghum P-450_(TYR).

Purified sorghum cytochrome P-450_(TYR) can be shown to form type Isubstrate binding spectra with tyrosine and N-hydroxytyrosine (comparesection 5.1.). Using P-450_(TYR) expressed in E. coli it can be shownthat in addition to tyrosine and N-hydroxytyrosine P-450_(TYR) is alsoable to form a type Ispectrum with p-hydroxyphenylacetaldehyde oxime,2-nitro-(p-hydroxyphenyl)ethane, p-hydroxyphenylacetonitrile as well asphenylalanine. The molar extinction coefficient E₄₂₀₋₃₉₀ for tyrosineand N-hydroxytyrosine as genuine substrates of P-450_(TYR) are 75.8 cm⁻¹mM⁻¹ and 64.6 cm⁻¹ mM⁻¹, respectively, whereas the extinctioncoefficients of the other compounds vary from 20-40 cm⁻¹ mM⁻¹.

Reconstitution experiments using phenylalanine as substrate do notresult in the production of the corresponding oxime. This indicates,that cytochrome P-450_(TYR) has a narrow substrate specificity withrespect to its enzymatic activity although it is able to bind manytyrosine analogues.

Administration of ¹⁴ C-tyrosine directly to E. coli cells expressingcytochrome P-450_(TYR) results in the production ofp-hydroxyphenylacetaldehyde oxime, indicating that E. coli is able toprovide the reducing equivalents for cytochrome P-450_(TYR).

The following oligonucleotides are used:

TYROL1d (SEQ ID NO: 23)

5'-CGG GAT CCA TAT GGC TCT GTT ATT AGC AGT TTT TCT GTC GTA CCT GGCCCG-3'

TYROL2 (SEQ ID NO: 20)

5'-GAC CGG CCG AAG CTT TAA TTA GAT GGA GAT GGA-3'

TYROL3 (SEQ ID NO: 21)

5'-AGT GGA TCC AGC GGA ATG CCG GCT T-3'

TYROL4 (SEQ ID NO: 22)

5'-CGT CAT GCT CTT CGG AA-3'

Example 6

Characterization of P-450_(Ox)

6.1. Substrate binding spectra of P450_(Ox)

Similiar experiments as reported in section 5.1 are carried out usingisolated cytochrome P-450_(Ox) with p-hydroxyphenylacetaldoxime andp-hydroxyphenylacetonitrile as substrate. Cytochrome P-450_(Ox) is foundto be multifunctional as P-450_(TYR). Isolated cytochrome P-450_(Ox)resembles the cytochrome P-450 reported to convert oximes to nitriles inrat liver microsomes (DeMaster et al, J. Org. Chem. 5074-5075, 1992).

6.2. Molecular weight and Amino acid sequence data

The molecular weight of P-450_(Ox) as determined by SDS-PAGE is 51 kD.

Amino acid sequences are obtained by automated Edman degradation. Theinternal polypeptides are obtained by trypsin digestion of the purifiedprotein and subsequent separation of peptides using reverse phase HPLC.

N-terminal sequence:

    --MDLADIPKQQRLMAGNALVV--                                   (SEQ ID NO: 12)

Additional peptide sequences:

    --ARLAEIFATII--                                            (SEQ ID NO:13)

    --EDFTVTTK--                                               (SEQ ID NO:14)

    --QYAALGSVFTVPII--                                         (SEQ ID NO: 15)

    --XXPFPI--                                                 (SEQ ID NO: 16)

6.3. Reconstitution of cytochrome P-450_(Ox) activity:

The reconstitution assay used for cytochrome P-450_(Ox) is similiar tothat used for cytochrome P-450_(TYR) described in section 5.3. A typicalassay contains 10 μl DLPC (10 mg/ml); 50 μl cytochrome P-450_(Ox) (24mg/ml); 50 μl NADPH-cytochrome P-450 oxidoreductase; 20 μl of either 10mM p-hydroxyphenylacetonitrile or p-hydroxyphenylacetaldehyde oxime; 10μl NADPH (25 mg/ml); and 60 μl potassium phosphate buffer (pH 7.9).

The reconstitution assay demonstrates that cytochrome P-450_(Ox)converts p-hydroxyphenylacetaldehyde oxime to p-hydroxymandelonitrile.The analytical procedures used are those described for cytochromeP-450_(TYR).

6.4. Inhibitory effect of antibodies against cytochrome P-450_(Ox)

The effect of antibodies raised against cytochrome P-450_(Ox) on thebiosynthetic activity is measured as the decrease in cyanide productionupon incubation of the sorghum microsomes withp-hydroxyphenylacetaldehyde oxime and p-hydroxybenzylcyanide assubstrates. The composition of the 150 μl total volume reaction mixturesis: microsomes containing 33 μg protein, 1,5 μmol substrate, 7,5 μmoltricine pH 8,0, 0,33 μmol NADPH, 0-255 μg antibodies and 0-255 μgreference immunoglobulin. The total amount of immunoglobulin in theassay is in each sample adjusted to 225 μg using purified immunoglobulinfrom a nonimmunized rabbit. The antibodies are preincubated with themicrosomes for 15 minute at 30° C. before substrate and NADPH are added.Subsequently the reaction is incubated at 30° C. for 30 minutes. Cyanideis determined by the Konig reaction (Konig, Z. Angew. Chem. 18:115,1905) using methodology described in Halkier and Moller, Plant Physiol.90:1552-1559, 1989. A value of A₆₈₀₋₅₈₅ =1,5 corresponds to 10 nmolescyanide. Protein concentration was determined using the method ofBradford (Bradford, Anal. Biochem. 72:248-254, 1976). A typical resultof such an inhibition experiment is shown in Table E.

                  TABLE E    ______________________________________    Substrate p-hydroxyphenylacetaldehyde oxime    μg antibody             0       15      30    60    120   225    ______________________________________    A.sub.680-585             1,00    0.96    0,92  0.87  0,88  0,66    inhibition             0%      4%      8%    13%   12%   34%    A.sub.680-555             1,07    1,03    1,03  0,88  0,87  0,70    inhibition             0%      4%      4%    18%   18%   35%    ______________________________________

The data show, that the antibody inhibits the reactions to the sameextent whichever substrate is added to the microsomal preparation.

Example 7

Induction of glucosinolate production in Tropaeolum majus.

Seeds of Tropaeolum majus L. cv Empress of India (DanskHavefroforsyning, Kolding, DK) are allowed to imbibe and germinate incomplete darkness for one week at 25° C. In vivo biosynthesisexperiments are performed wherein 1 μCi of the tracer ¹⁴ C-labelledphenylalanine is administered to excised dark-grown seedlings for 24hours followed by boiling of the plant material in 90% methanol andanalysis of the extracts by HPLC as described by Lykkesfeldt and Moller,1993. Prior to administration of the tracer to the excised seedling orleaf, the intact plant is subjected to a potential inducer for 24 hours.Administration of 10 mM phenylalanine or 2% ethanol to the vermiculatein which the etiolated seedlings are grown results in a threefoldincrease in glucosinolate production as compared to control experimentswith water. Spraying with 100 μM jasmonic acid followed by incubationfor 24 hours results in a fivefold induction in etiolated seedlings andgreen leaves.

Example 8

Preparation of biosynthetically active microsomes fromglucosinolate-producing plant meterial

The biosynthetic pathways of glucosinolates, and cyanogenic glucosidesshare homology by having amino acids as precursors and oximes asintermediates. The assignment of amino acids and oximes as precursorsand intermediates in the glucosinolate biosynthetic pathway is based onin vivo experiments demonstrating that these compounds are efficientprecursors for glucosinolates. In vitro biosynthetic studies havehitherto not been possible due to the detrimental effect of thedegradation products of glucosinolates on enzyme activities. Thedegradation products are formed upon disruption of the cellularstructure. In the disrupted tissue, the glucosinolate-degrading enzymemyrosinase gets in contact with the glucosinolates resulting in thegeneration of isothiocyanates inactivating the enzymes. We demonstratethat microsomal preparations isolated form one week old plants of eitherSinapis alba or Trapaeolum majus are able to convert tyrosine andphenylalanine, respectively, to the corresponding oximes. Theenzymatically active microsomal preparations are obtained by using anisolation buffer fortified with 100 mM ascorbic acid known to inhibitthe activity of myrosinase and by inducing the glucosinolate-producingenzyme system prior to the preparation of microsomes. Theglucosinolate-producing enzyme systems are induced by taking 7-days-olddark-grown Sinapis plants or 3-4 weeks old light-grown Tropaeolum plantsand placing them in the light for 3 days. During this three day period,the young plants are sprayed with 50 μM jasmonic acid once a day. After3 days of induction, the plants are harvested and microsomes areprepared as described in section 5.1, except that the homogenisationbuffer consists of 250 mM Tricine pH 7.9, 250 mM sucrose, 50 mM sodiumbisulfite, 100 mM ascrobic acid, 4 mM DDT, 2 mM EDTA, 1 mM PMSF, and 5mg/ml BSA. The microsomal preparation is dialysed against homogenizationbuffer for 1 hour, followed by dialysis against 50 mM Tricine pH 7.9 and2 mM DTT for another hour.

Example 9

In vitro biosynthesis of oxime by extracts from glucosinolate-containingplants

The microsomal reaction mixture consists of 80 μl microsomes (10 mgprotein per ml), 10 μl ¹⁴ C-phenylalanine (0.5 μCi, 464 mCi/mmol,Amersham) or ¹⁴ C-tyrosine (0.5 μCi, 450 mCi/mmol, Amersham) and 10 μlNADPH (75 mg/ml). The reaction mixtures are incubated for 1 hour at 37°C. At the end of the incubation period, the reaction mixtures areextracted with 1500 μl ethyl acetate. The ethyl acetate phase isevaporated to dryness, redissolved in a small volume and analyzed. Theproduction of oximes in the microsomal reaction mixtures can bedemonstrated by thin layer chromatography as well as by HPLC analysis asdescribed in section 5.3.

Example 10

Involvement of cytochrome P450-dependent monooxygenases in theglucosinolate pathway

Based on the similarity between the first part of the biosyntheticpathways of glucosinolates and cyanogenic glucosides, it was anticipatedthat the conversion of amino acid to oxime in the glucosinolate pathwayis catalyzed by a multifunctional cytochrome P450 monooxygenasehomologous to P450_(I) in the cyanogenic glucoside pathway. In vivoexperiments, where radioactively labelled phenylalanine is administeredto etiolated tropaeolum seedlings in the presence and absence of 1 mM ofthe cytochrome P450 inhibitors enilketoconazol and tetcyclacisdemonstrate that cytochrome P450 inhibitors cause a reduction ofglucosinolate without causing a reduction in the uptake of phenylalanineas measured by ethanol extraction of the plant material. This indicatesthat the biosynthesis of glucosinolates is dependent on cytochrome P450.

Direct demonstration of the involvment of cytochrome P450 inglucosinolate biosynthesis can be obtained using the in vitro microsomalenzyme system from tropaeolum to demonstrate photoreversible carbonmonoxide inhibition of oxime production. The microsomal reactionmixtures are incubated using different experimental conditions. Thereaction mixtures are analyzed by HPLC.

    ______________________________________    Experimental Condition                   % inhibition of oxime production    ______________________________________    O.sub.2 without light                    0    O.sub.2 with light                   11    CO/O.sub.2 without light                   65    CO/O.sub.2 with light                   23    ______________________________________

The possibility to reactivate the microsomal enzyme system uponirradiation with 450 nm light shows, that the conversion ofphenylalanine to the corresponding oxime in the biosynthetic pathway ofglucosinolate is dependent on cytochrome P450.

Example 11

Toxicity of cyanogenic glycosides for insects.

Insects or insect larvae are fed on a diet containing added cyanogenicglycoside, a diet containing added cyanogenic glycoside, and callus, ora diet supplemented with the supernatant of callus ground-up in thepresence of the cyanogenic glycoside. Mortality is compared to themortality of insects or insect larvae fed on the diet only.

Example 12

Activity of Amygdalin on larval mortality of Western Corn Root Worm(WCRW):

WCRW larvae are fed on a diet with added amygdalin, a diet with addedamygdalin and Black Mexican Sweet (BMS) callus or on a diet supplementedwith the supernatant of BMS-callus ground-up in the presence ofamygdalin. Larval mortality is compared to the mortality of larvae fedon the diet only.

The results show that amygdalin is lethal in the presence of BMS-calluswith an LC₅₀ of ˜1 mg/ml, and that it is lethal at 2 mg/ml in theabsence of BMS-callus. There is significantly less lethality atamygdalin concentrations of less than 1 mg/ml when BMS-callus is absent.

Example 13

Activity of Dhurrin on larval mortality of Western Corn Root Worm(WCRW):

The activity of dhurrin on larval mortality of WCRW was determined asdescribed for amygdalin in example 12.

The results show that the LC₅₀ of dhurrin is 368 μg/ml with 95%confidence limits of 0.28-0.48 μg/ml. The slope of the regression lineis 2.5.

Example 14

Transfection of maize by direct Bombarding of Immature Zygotic Embryosand Isolation of Transformed Callus with the Use of Phosphinothricin asa selection agent.

Immature embryos are obtained approximately 14 days afterself-pollination. The immature zygotic embryos are divided amongdifferent target plates containing medium capable of inducing andsupporting embryogenic callus formation at 36 immature embryos perplate. The immature zygotic embryos are bombarded with plasmids encodinga cytochrome P-450 monooxygenase and a chimeric gene coding forresistance to phosphinothricin using the PDS-1000/He device from DuPont.The plasmids are precipitated onto 1 μm gold particles essentiallyaccording DuPont's procedure. Each target plate is shot one time withthe plasmid and gold preparation and phosphinothricin is used to selecttransformed cells in vitro. Selection is applied at 3 mg/l one day afterbombardment and maintained for a total of 12 weeks. The embryogeniccallus so obtained is regenerated in the absence of the selection agentphosphinothricin. The regenerated plants are tested for their resistanceto insects, acarids or nematodes.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be obvious that certain changes and modificationsmay be practiced within the scope of the appended claims.

                  TABLE A    ______________________________________    PLANT CLASSIFICATION ACCORDING TO USE    ______________________________________    CEREALS    Monocot    Avena nuda (chinensis)                    Chines naked oat    A. sativa       Common oats    Eleusine coracan                    African millet    Eragrostis tef  Tef grass    Fagopyrum esculentum                    Buckwheat    F. tataricum    Rye buckwheat    Hordeum distichum                    Two-row barley    H. vulgare      Barley    Oryza sativa    Rice    Panicum italicium                    Italian millet    P. miliaceum    Broomcorn millet    Pennisetum glaucum                    Spiked millet    P. spicatum (americanum)                    Perl millet    Secale cereale  Rye    Sorghum vulgare Grain sorghums    X Triticosecale Triticale    Triticum aestivum                    Common wheat    T. dicoccum     Emmer    T. durum        Abyssinian hard wheat    T. monococcum   Einkorn wheat    Zea mays        Corn, sweet corn    Dicot    Amaranthus paniculatus                    Rispenfuchsschwanz    Fagopyrum esculentum                    Buchweizen    F. tataricum    PROTEIN CROPS    Dicot    Arachis hypogea Groundnut, peanut    Cajanus indicus Pigeon pea    Cicer arietinum Chickpea    Dolichos lablab Hyacinth bean    Glycine gracilis                    Manchurian Soya    G. max          Soyabean    G. ussuriensis  Wild soya    Lathyrus sativus                    Grass pea    Lens culinaris  Lentil    Mucuna pruriens Cowitch, Florida velvet bean    Phaseolus acutifolius                    Tepary bean    P. aureus       Mung, green gram    P. lunatus      Lima bean, Sieva    P. coccineus    Scarlet runner bean    (multiflorus)    P. mungo        Black gram    P. vulgaris     French, common, kidney or dwarf bean    Vicia faba      Horse bean, broad bean    Vigna angularis Adzuki bean    V. sesquipedalis                    Asparagus (yard-long bean)    V. sinensis     Cowpea    FRUIT CROPS    Dicot    Amygdalus communis                    Almond    Ananas comosus  Pineapple    Artocarpus communis                    Breadfruit    Carica papaya   Papaya    Citrullus vulgaris                    Watermelon    Citrus grandis  Pummelo    C. medica       Citron, lemon    C. nobilis      Tangerine    C. reticulata   Mandarin    C. sinensis     Orange    Cydonia oblonga Quince    Diospyros kaki  Japanese persimmon    Ficus carica    Fig    Fragaria chiloensis                    Wild strawberry    F. virginiana   Strawberry    Litchi chinensis                    Litchi    Malus asiatica  Chines apple    M. pumila       Appple    Mangifera indica                    Mango    Morus rubra     Red mulberry    Musa cavendishii                    Banana    M. paradisiaca  Banana    Passiflora edulis                    Passion fruit, purple granadilla    P. ligularis    Passion flower    Persea americana                    Avocado pear    Phoenix dactylifera                    Date palm    Prunus armeniaca                    Apricot    P. avium        Sweet cherry, mazzard    P. cerasifera   Cherry plum    (divaricata)    P. cerasus      Cherry    P. domestica    European plum or prune    P. maheleb      Maheleb cherry    P. persica      Peach and nectarine    P. pseudocerasus                    Cherry    P. salicinia    Japanese peach    P. serotina     Wild black cherry    Psidium guajava Guava    Punica granatum Pomegranate    Pyrus communis  Pear    P. ussuriensis  Chinese pear    Ribes grossularia                    Gooseberry    R. nigrum       Black currant    R. rubrum       Red and white currant    Rubus idaeus    European raspberry    R. strigosus    American raspberry    Tamarindus indica                    Tamarind    Vaccinium angustifolium                    Sugarberry    V. ashei        Rabbiteye blueberry    V. corymbosum   Highbush blueberry    V. myrtilloides Canada blueberry    V. oxycoccos    Cranberry    Viburnum trilobum                    American cranberry bush    Vitris labrusca Fox grape    V. vinifera     Grape    VEGETABLES AND TUBERS    Monocot    Allium ascalonicum                    Shallot, breen onion    A. cepa         Onion    A. chinense     Onion    A. fistulosum   Welch onion    A. porrum       Leek    A. sativum      Garlic    A. schoenoprasum                    Chives    Asparagus officinalis                    Asparagus (var. attilis)    Zea mays        sweet corn    Dicot    Amoracia lapathifolia                    Horseradish    Apium graveolens                    Celery    Arabidopsis thaliana                    Common wall cress    Beta vulgaris   Sugar, mangold or garden beet    Brassica alboglabra                    Chinese kale    B. campestris   Turnip rape    B. carinata     Ambyssian mustard    B. cernea       Karashina    B. chinensis    Chinese mustard or pak-choi    B. hirta        White mustard    B. juncea       Pai, brown mustard, Indian mustard    B. kaber        Charlock    B. napobrassica Swede or rutabaga    B. napus        Rape, oil rape, kale    B. nigra        Black mustard    B. oleracea     Cole, kale, collards, brussels sprouts,                    cauliflower, cabbage, kohlrabi, broccoli    B. pekinensis   Chines cabbage or celery cabbage    B. rapa         Turnip    Cajanus cajan (indicus)                    Pigeon pea    Canavalia ensiformis                    Jack bean    Canna edulis    Edible canna    Capsicum annuum Common cultivated pepper    C. chinense     Pepper    C. frutescens   Cayenne pepper    C. pendulum     Pepper    C. pubescens    Pepper    Cichorium endivia                    Endive    C. intybus      Chicory    Colocasia antiquorum                    Taro    Crambe maritima Sea kale    Cucumis melo    Melon, cantaloupe    C. sativus      Cucumber    Cucurbita ficifolia                    Malabar gourd    C. foetidissima Calabazilla, buffalo gourd    C. maxima       Pumpkin    C. moschata     Winter pumpkin    C. pepo         Summer squash, vegetable marrow    Cynara scolymus Globe artochoke    Daucus carota   Carrot    Dioscorea alata Yarn    D. batatas      Chines yarn    D. cavennensis  Attoto yam    Eruca sativa Mill.                    Rocket salad, rocket or roquette    Ipomea batatas  Sweet potato    Lactuca sativa  Lettuce    Lepidium sativum                    Garden cress    Lycopersicon cerasiforme                    Cherry tomato    L. esculentum   Tomato    Manihot esculenta                    Manioc, cassava    Nasturtium officinale                    Water cress    Pastinaca sative                    Parsnip    Petroselinum crispum                    Parsley    (sativum)    Physalis peruviana                    Ground cherry    Pisum sativum   Pea    Raphanus sativus                    Radish    Rheum officinale                    Rhubarb    R. rhaponticum  English rhabarb    Scorzonera hispanica                    Black salsify    Sechium edule   Chayote    Solanum andigenum                    Andean potato    S. melongena    Eggplant    S. muricatum    Pepino    S. phureja      Potato    S. tuberosum    Common potato    Psinacia oleracea                    Spinach    NUTS    Dicot    Anacardium occidentale                    Cashew    Arachis hypogaea                    Peanut    Carya illinoinensis                    Pecan    C. ovata        Shagbark hickory    Castanea sativa Chestnut    Covos nucifera  coconut palm    Corylus americana                    American hazel, filbert    C. aveliana     European hazel, cobnut    Juglans nigra   Black walnut    J. regia        English walnut    J. sinensis     Walnut    Litchi chinensis                    Litchi    Macadamia integrifolia                    Queensland nut    Pistacia vera   Pistachio nut    Prunus amygdalus                    Almond    OIL CROPS    Monocot    Zea mays        Corn    Dicot    Aleurites cordata                    Tung, China wood oil    A. moluccana (triloba)                    Candlenut    Arachis hypogea Ground nut, penut    brassica campestris                    Rapeseed oil, canola oil    B. napus        Rapeseed oil, canona oil    Cannabis sativa Hampseed oil    Carthamus tinctorius                    Safflower oil    Cocos nucifera  Coconut palm    Elaeis guineensis                    Oil palm    Glycine gracilus                    Manch, soya    G. max          Soybean    G. ussuriensis  Wild soya    Cossypium hirsutum                    Cottonseed oil    Helianthus annus                    Sunflower    Linum usitatissimum                    Flax    Olea europaea   Olive    Papaver somniferum                    Poppy seed    Ricinus communis                    Castor bean    Sesamum indicum Sesame    SUGAR CROPS    Monocot    Saccharum officinarum                    Sugarcane    (officinarum x    spontaneum)    S. robustum    S. sinense      Sugarcane    S. spontaneum   Kans grass    Sorghum dochna  Sorgo syrup, sugar sorghun    Dicot    Acer saccharum  Sugar maple    Beta vulgaris   Sugar or mangold beet    FORAGE AND TURF GRASSES    Monocot    Agropyron cristatum                    Crested wheatgrass    A. desertorum   Crested wheatgrass    A. elongatum    Tall wheatgrass    A. intermedium  Intermediate wheatgrass    A. smithii      Western wheatgrass    A. spicatum     Blue bunch wheatgrass    A. trachycaulum Slender wheatgrass    A. trichophorum Pubescen wheatgrass    Alopecurus pratensis                    Meadow foxtail    Andropogon gerardi                    Big bluestem    Arrhenatherum elatius                    Tall oat grass    Bothrichloa barbinodis                    Cane blestem    B. ischaemum    King ranch bluestem    B. saccharoides Silver bluestem    Bouteloua curipendula                    Side oats grama    B. eriopoda     Black grama    B. gracilis     Blue grama    Bromus erectus  Upright brome    B. inermis      Smooth brome    B. riparius     Meadow brome    Cenchrus ciliaris                    Buffel grass    Chloris gayana  Rhodes grass    Cymbopogon nardus                    Citronella grass    Cynodon dactylon                    Bermuda grass    Dactylis glomerata                    Cocksfoot    Dichanthium annulatum                    Kleberg bluestem    D. aristatum    Angleton bluestem    D. sericeum     Silky bluestem    Digitaria decumbens                    Pangola grass    D. smutsii    Elymus angustus Altai wild rye    E. junceus      Russian wild rye    Eragrostis curvula                    Weeping love grass    Festuca arundinacea                    Tall fescue    F. ovina        Sheeps fescue    F. pratensis    Meadow fescue    F. rubra        Red fescue    Lolium multiflorum                    Italian ryegrass    L. perenne      Perennial ryegrass    Panicum maximum Guinea grass    P. purpurascens Para grass    P. virgatum     Switchgrass    Paspalum dilatatum                    Dallis grass, large water grass    P. notatum      Bahia grass    Pennisetum clandestinum                    Kikuyu grass    P. purpureum    Dry napier grass    Phalaris arundinacea                    Reed canary grass    Phleum bertolinii                    Timothy    P. pratense     Timothy    Poa fendleriana Mutton grass    P. nemoralis    Wood meadow grass    P. pratensis    Kentucky bluegrass    Setaria sphacelata                    Rhodesian timothy    Sorghastrum nutans                    Indian grass    Sorghum halepense                    Johnson grass    S. sudanense    Sudan grass    Sorghum vulgare Great millet    FORAGE LEGUMES    Dicot    Coronilla varia Crown vetch    Crotalaria juncea                    Sun hemp    Lespedeza stipulacea                    Korean lespedeza    L. striata      Common lespedeza    L. sericea    Lotus corniculatus                    Birdsfoot trefoil    L. uliginosus    Lupinus albus   Wolf bean, white lupin    L. angustifolius                    Blue lupin    L. luteus       European yellow lupin    L. mutabilis    South American lupin    Medicago arabica                    Spotted burr-clover    M. arborea      Tree alfalfa    M. falcata      Yellow lucerne    M. hispida      California burr-clover    M. sativa       Alfalfa    M. tribuloides  Barrel medic    Melilotus albus White sweet clover    M. officinalis  Yellow sweet clover    Onobrychis viciifolia                    Sainfoin    Ornithopus sativus                    Serradella    Pueraria thunbergiana                    Kudzu vine    Trifolium alexandrinum                    Egyptian clover    T. augustifolium                    Fineleaf clover    T. diffusum     Rose clover    T. hybridum     Alsike clover    T. incarnatum   Crimson clover    T. ingrescens   Ball clover    T. pratense     Red clover    T. repens       White clover    T. resupinatum  Persian clover    T. subterraneum Subterranean clover    Trigonella foenumgraecum                    Fenugreek    Vicia sative    Common vetch    V. villosa      Hairy vetch    V. atropurpurea Purple vetch    V. angustifolia Narrowleaf vetch    V. dasycarpa    Wooly pod vetch    V. ervilia      Monantha (bitter) vetch    V. pannonica    Hungarian vetch    V. calcarata    Bard vetch    FIBER PLANTS AND WOODY PLANTS    Monocot    Bambusa vulgaris                    Bamboo    Dicot    Agave sisalana  Sisal hemp    Boehmeria nivea Rhea fiber, ramie    Cannabis indica Hemp    C. sativa       Hemp    Ceiba pentandra Silk cotton tree, kapok tree    Corchorus mucronata                    Hemp    (striata)    Gossypium arboreum                    Tree cotton    G. barbadense   Egyptian cotton    G. herbaceum    Cotton    G. hirsutum     Upland cotton    G. nanking      Oriental cotton    Linum angustifolium                    Wild flax    L. usitatissimum                    Flax    Musa textiles   Manila hemp, abaca    DRUG CROPS    Dicot    Angelica archangelica                    Angelica    Chrysanthemum cinerariifolium                    Palm pyrethrum    Camellia sinensis                    Chinese tea    C. coccineum    Pyrethrum    Coffea arabica  Coffee    C. canephora    Quillow coffee    Cola acuminata  Kola nut    Nicotiana rustica                    Tobacco    N. tabacum      Tobacco    Papaver dubium  Poppy    P. somniferum   Opium poppy    Theobroma cacao cocoa    SPICES AND FLAVORINGS    Monocot    Vanilla fragrans                    Vanilla    Dicot    Artemisa dracunculus                    Tarragon    Cinnamomum zeylanicum                    Cinnamon tree    Hibiscus esculentus                    Okra    Salvia officinalis                    Sage    Thymus vulgaris Thyme    Pimpinella anisum                    Anise    Mentha arvensis Menthol    M. piperita     Peppermint    M. viridis      Spearmint    Coriandrum sativum                    Coriander    ______________________________________

                  TABLE B    ______________________________________    REPRESENTATIVE PLANT PESTS    ______________________________________    Coleoptera:    Diabrotica, Melanotus, Agriotes, Limonius, Dalopius, Eleodes,    Chaetocnema, Macrodactylus, Sphenophorus, Sitophilus, Lisorhoptrus,    Oulema, Rhyzopertha, Prostephanus, Phyllophage, Cyclocephala, Popillia,    Anthonomus, Zabrotes, Leptinotarsa    Lepidoptera:    Heliothis, Ostrinia, Diatraea, Elasmopalpus, Papaipema, Agrotis,    Loxagrotis, Euxoa, Peridroma saucia, Chorizagrotis, Spodoptera,    Pseudaletia, Chilo, Busseola, Sesamia, Eldana, Maliarpha, Scirpophaga,    Duataea, Rupela, Sitotroga cerealella, Sitroga, Plodia interpunctella,    Crambus, Mythimna, Nola, Pectinophora, Acontia, Trichoplusia,    Anticarsia, Pseudoplusia, Manduca, Leptinotarsa, Lema    Thysanoptera:    Frankliniella, Anaphothrips, Hercothrips, Stenothrips    Homoptera:    Dalbulus, Cicadulina, Rhopalosiphum, Melanaphis, Anuraphis, Prosapia,    Nilaparvata, Sogatella, Laodelphax, Sogatodes, Nephotettix, Reciian,    Cofana, Empoasca, Poophilus, Schizaphis, Sipha, Paratrioza, Empoasca,    Ophilia. Scleroracus, Macrosteles, Circulifer, Aceratagallia, Agallia,    Myzus, Macrosiphum, Aphis    Diptera:    Delia platura, Euxesta, Diopsis, Atherigona, Hydrellia, Orseolia,    Chironomus, Contarinia    Orthoptera:    Melanoplus, Schistocerca, Sphenarium, Aneolamia    Isoptera:    Microtermes, Macrotermes, Allodontermes, Odontotermes    Heteroptera:    Nezara, Acrosternum, Euschistus, Blissus    Acarina:    Tetranychus, Paratetranychus, Oligonychus    ______________________________________

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    ______________________________________    EP-240 208-A2 WO 89/05852  US 5,023,179    EP-306, 139-A WO 89/07647  US 5,231,020    EP 332 104    WO 91/13992    EP-452 269-A2 WO 93/07278    EP-458 367 A1 WO 92/09696                  WO 90/11682    ______________________________________

    __________________________________________________________________________    SEQUENCE LISTING    (1) GENERAL INFORMATION:    (iii) NUMBER OF SEQUENCES: 24    (2) INFORMATION FOR SEQ ID NO: 1:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 2143 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: double    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA to mRNA    (iii) HYPOTHETICAL: NO    (iii) ANTI-SENSE: NO    (vi) ORIGINAL SOURCE:    (A) ORGANISM: Sorghum bicolor    (vii) IMMEDIATE SOURCE:    (B) CLONE: P-450-Tyr    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:    CCGGCTAGCTAGCTCATCGGGTGATCGATCAGTGAGCTCTCTCTTTGGCCTAGCTAGCTG60    CTAGCAGTGCAGGTAGCCAATCAAAGCAGAAGAACTCGATCGATCGATCATCACGATCGC120    TGCTAGCTAGCTAGCTGCTCGCTCTCACACTAGCTACGTGTTTTTGTTAATTTGATATAT180    ATATATAATGGCGACAATGGAGGTAGAGGCCGCGGCCGCCACGGTGCTGGCCGCGCCCTT240    GCTGTCCTCCTCCGCGATCCTCAAACTGCTGCTATTCGTAGTGACGCTCTCGTACCTGGC300    CCGAGCCCTGAGGCGGCCACGCAAAAGCACCACCAAGTGCAGCAGCACAACGTGCGCCTC360    GCCCCCGGCCGGCGTTGGCAACCCGCCGCTCCCACCGGGTCCCGTGCCGTGGCCCGTCGT420    CGGCAACCTGCCGGAGATGCTGCTGAACAAGCCGGCATTCCGCTGGATCCACCAGATGAT480    GCGCGAGATGGGCACGGACATCGCCTGCGTCAAGCTTGGCGGCGTCCACGTCGTGTCCAT540    CACCTGCCCGGAGATCGCGCGGGAGGTGCTCCGGAAGCAGGACGCCAACTTCATATCCCG600    CCCGCTCACCTTCGCCTCCGAGACGTTCAGCGGCGGGTACCGGAACGCCGTGCTCTCGCC660    CTACGGCGACCAGTGGAAGAAGATGCGCCGCGTCCTCACCTCCGAGATCATCTGCCCGTC720    CCGCCACGCCTGGCTCCACGACAAGCGCACCGACGAGGCCGACAACCTCACCCGCTACGT780    CTACAACCTCGCCACCAAAGCCGCCACCGGCGACGTCGCCGTCGACGTCAGGCACGTCGC840    TCGTCACTATTGCGGCAACGTTATCCGCCGCCTCATGTTCAACAGGCGCTACTTCGGCGA900    GCCCCAGGCTGACGGCGGTCCGGGGCCGATGGAGGTGCTGCATATGGACGCCGTGTTCAC960    CTCCCTCGGCCTCCTCTACGCCTTCTGCGTCTCCGACTACCTCCCCTGGCTGCGGGGCCT1020    CGACCTCGACGGCCACGAGAAGATCGTCAAGGAGGCTAACGTGGCGGTGAACAGGCTCCA1080    CGACACGGTCATCGACGACCGGTGGAGGCAGTGGAAGAGCGGCGAGCGGCAGGAGATGGA1140    GGACTTCCTGGATGTGCTCATCACTCTCAAGGACGCCCAGGGCAACCCGCTGCTGACCAT1200    CGAGGAGGTCAAAGCGCAGTCACAGGACATCACGTTCGCGGCGGTGGACAACCCGTCGAA1260    CGCCGTGGAGTGGGCGCTGGCAGAGATGGTGAACAACCCGGAGGTGATGGCGAAGGCGAT1320    GGAGGAGCTGGACCGCGTCGTCGGACGGGAGAGGCTAGTGCAGGAGTCGGACATTCCGAA1380    GCTCAACTACGTGAAGGCCTGCATCCGGGAGGCTTTCCGTCTGCACCCGGTGGCGCCCTT1440    CAACGTGCCCCACGTCGCGCTCGCCGACACCACCATCGCCGGCTACCGCGTTCCCAAGGG1500    CAGCCACGTGATCCTGAGCCGCACGGGGCTGGGCCGCAACCCGCGCGTGTGGGACGAGCC1560    CCTGCGCTTCTACCCGGACCGACACCTCGCCACCGCCGCGTCCGACGTCGCGCTCACCGA1620    GAACGACCTGCGGTTCATCTCCTTCAGCACCGGCCGCCGCGGCTGCATCGCCGCGTCGCT1680    CGGCACCGCCATGAGCGTCATGCTCTTCGGAAGGCTCCTGCAGGGGTTCACCTGGAGCAA1740    GCCCGCCGGGGTGGAGGCCGTGGACCTCAGCGAGTCCAAGAGCGACACCTTCATGGCCAC1800    CCCGCTGGTGCTGCACGCTGAGCCCAGGCTGCCGGCGCACCTCTACCCGTCCATCTCCAT1860    CTGATTAAACGTACGGCCGGTCGTCATTATATTGTATGCATATAATTAAAGACGAGCGAG1920    CCTGCTGGTCACACTTGCATTGCATGTATCATCAGCAGGGGGCTATGCAATAAGTTTTTT1980    TTTTCCGCGCTTGATTTCGTGGTGCTGTGCGTATTCTGCGCACACCGACTGTACGTACGA2040    CGGCGTTCAGCTTTGTATTGTACCGAGTTAAAAAGTATTATTATTATTATCATCGACAAT2100    AATAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA2143    (2) INFORMATION FOR SEQ ID NO: 2:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 558 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (iii) HYPOTHETICAL: NO    (iii) ANTI-SENSE: NO    (vi) ORIGINAL SOURCE:    (A) ORGANISM: Sorghum bicolor    (vii) IMMEDIATE SOURCE:    (B) CLONE: P-450-Tyr    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:    MetAlaThrMetGluValGluAlaAlaAlaAlaThrValLeuAlaAla    151015    ProLeuLeuSerSerSerAlaIleLeuLysLeuLeuLeuPheValVal    202530    ThrLeuSerTyrLeuAlaArgAlaLeuArgArgProArgLysSerThr    354045    ThrLysCysSerSerThrThrCysAlaSerProProAlaGlyValGly    505560    AsnProProLeuProProGlyProValProTrpProValValGlyAsn    65707580    LeuProGluMetLeuLeuAsnLysProAlaPheArgTrpIleHisGln    859095    MetMetArgGluMetGlyThrAspIleAlaCysValLysLeuGlyGly    100105110    ValHisValValSerIleThrCysProGluIleAlaArgGluValLeu    115120125    ArgLysGlnAspAlaAsnPheIleSerArgProLeuThrPheAlaSer    130135140    GluThrPheSerGlyGlyTyrArgAsnAlaValLeuSerProTyrGly    145150155160    AspGlnTrpLysLysMetArgArgValLeuThrSerGluIleIleCys    165170175    ProSerArgHisAlaTrpLeuHisAspLysArgThrAspGluAlaAsp    180185190    AsnLeuThrArgTyrValTyrAsnLeuAlaThrLysAlaAlaThrGly    195200205    AspValAlaValAspValArgHisValAlaArgHisTyrCysGlyAsn    210215220    ValIleArgArgLeuMetPheAsnArgArgTyrPheGlyGluProGln    225230235240    AlaAspGlyGlyProGlyProMetGluValLeuHisMetAspAlaVal    245250255    PheThrSerLeuGlyLeuLeuTyrAlaPheCysValSerAspTyrLeu    260265270    ProTrpLeuArgGlyLeuAspLeuAspGlyHisGluLysIleValLys    275280285    GluAlaAsnValAlaValAsnArgLeuHisAspThrValIleAspAsp    290295300    ArgTrpArgGlnTrpLysSerGlyGluArgGlnGluMetGluAspPhe    305310315320    LeuAspValLeuIleThrLeuLysAspAlaGlnGlyAsnProLeuLeu    325330335    ThrIleGluGluValLysAlaGlnSerGlnAspIleThrPheAlaAla    340345350    ValAspAsnProSerAsnAlaValGluTrpAlaLeuAlaGluMetVal    355360365    AsnAsnProGluValMetAlaLysAlaMetGluGluLeuAspArgVal    370375380    ValGlyArgGluArgLeuValGlnGluSerAspIleProLysLeuAsn    385390395400    TyrValLysAlaCysIleArgGluAlaPheArgLeuHisProValAla    405410415    ProPheAsnValProHisValAlaLeuAlaAspThrThrIleAlaGly    420425430    TyrArgValProLysGlySerHisValIleLeuSerArgThrGlyLeu    435440445    GlyArgAsnProArgValTrpAspGluProLeuArgPheTyrProAsp    450455460    ArgHisLeuAlaThrAlaAlaSerAspValAlaLeuThrGluAsnAsp    465470475480    LeuArgPheIleSerPheSerThrGlyArgArgGlyCysIleAlaAla    485490495    SerLeuGlyThrAlaMetSerValMetLeuPheGlyArgLeuLeuGln    500505510    GlyPheThrTrpSerLysProAlaGlyValGluAlaValAspLeuSer    515520525    GluSerLysSerAspThrPheMetAlaThrProLeuValLeuHisAla    530535540    GluProArgLeuProAlaHisLeuTyrProSerIleSerIle    545550555    (2) INFORMATION FOR SEQ ID NO: 3:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 17 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (iii) HYPOTHETICAL: NO    (iii) ANTI-SENSE: NO    (v) FRAGMENT TYPE: N-terminal    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:    MetAlaThrMetGluValGluAlaAlaAlaAlaThrValLeuAlaAla    151015    Pro    (2) INFORMATION FOR SEQ ID NO: 4:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 7 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (iii) HYPOTHETICAL: NO    (iii) ANTI-SENSE: NO    (v) FRAGMENT TYPE: internal    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:    ValTrpAspGluProLeuArg    15    (2) INFORMATION FOR SEQ ID NO: 5:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 8 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (iii) HYPOTHETICAL: NO    (iii) ANTI-SENSE: NO    (v) FRAGMENT TYPE: internal    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:    TyrValTyrAsnLeuAlaThrLys    15    (2) INFORMATION FOR SEQ ID NO: 6:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 16 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (iii) HYPOTHETICAL: NO    (iii) ANTI-SENSE: NO    (v) FRAGMENT TYPE: internal    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:    SerAspThrPheMetAlaThrProLeuValSerSerAlaGluProArg    151015    (2) INFORMATION FOR SEQ ID NO: 7:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 34 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (iii) HYPOTHETICAL: NO    (iii) ANTI-SENSE: NO    (v) FRAGMENT TYPE: internal    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7:    AlaGlnSerGlnAspIleThrPheAlaAlaValAspAsnProSerAsn    151015    AlaValGluXaaAlaLeuAlaGluMetValAsnAsnProGluValMet    202530    AlaLys    (2) INFORMATION FOR SEQ ID NO: 8:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 13 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (iii) HYPOTHETICAL: NO    (iii) ANTI-SENSE: NO    (v) FRAGMENT TYPE: internal    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8:    AlaGlnGlyAsnProLeuLeuThrIleGluGluValLys    1510    (2) INFORMATION FOR SEQ ID NO: 9:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 9 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (iii) HYPOTHETICAL: NO    (iii) ANTI-SENSE: NO    (v) FRAGMENT TYPE: internal    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9:    LeuValGlnGluSerAspIleProLys    15    (2) INFORMATION FOR SEQ ID NO: 10:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 6 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (iii) HYPOTHETICAL: NO    (iii) ANTI-SENSE: NO    (v) FRAGMENT TYPE: internal    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10:    IleSerPheSerThrGly    15    (2) INFORMATION FOR SEQ ID NO: 11:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 12 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (iii) HYPOTHETICAL: NO    (iii) ANTI-SENSE: NO    (v) FRAGMENT TYPE: internal    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11:    LeuProAlaHisLeuTyrProSerIleSerLeuHis    1510    (2) INFORMATION FOR SEQ ID NO: 12:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 20 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (iii) HYPOTHETICAL: NO    (iii) ANTI-SENSE: NO    (v) FRAGMENT TYPE: N-terminal    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12:    MetAspLeuAlaAspIleProLysGlnGlnArgLeuMetAlaGlyAsn    151015    AlaLeuValVal    20    (2) INFORMATION FOR SEQ ID NO: 13:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 11 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (iii) HYPOTHETICAL: NO    (iii) ANTI-SENSE: NO    (v) FRAGMENT TYPE: internal    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13:    AlaArgLeuAlaGluIlePheAlaThrIleIle    1510    (2) INFORMATION FOR SEQ ID NO: 14:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 8 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (iii) HYPOTHETICAL: NO    (iii) ANTI-SENSE: NO    (v) FRAGMENT TYPE: internal    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14:    GluAspPheThrValThrThrLys    15    (2) INFORMATION FOR SEQ ID NO: 15:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 14 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (iii) HYPOTHETICAL: NO    (iii) ANTI-SENSE: NO    (v) FRAGMENT TYPE: internal    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15:    GlnTyrAlaAlaLeuGlySerValPheThrValProIleIle    1510    (2) INFORMATION FOR SEQ ID NO: 16:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 6 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (iii) HYPOTHETICAL: NO    (iii) ANTI-SENSE: NO    (v) FRAGMENT TYPE: internal    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16:    XaaXaaProPheProIle    15    (2) INFORMATION FOR SEQ ID NO: 17:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 17 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (iii) HYPOTHETICAL: NO    (vii) IMMEDIATE SOURCE:    (B) CLONE: Oligonucleotide specifying AA sequence MEVEAA    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 17:    ATGGARGTNGARGCNGC17    (2) INFORMATION FOR SEQ ID NO: 18:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 17 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (iii) HYPOTHETICAL: NO    (vii) IMMEDIATE SOURCE:    (B) CLONE: Oligonucleotide specifying AA sequence DFTMAT    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 18:    GAYACNTTYATGGCNAC17    (2) INFORMATION FOR SEQ ID NO: 19:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 42 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (iii) HYPOTHETICAL: NO    (vii) IMMEDIATE SOURCE:    (B) CLONE: TYROL1b    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 19:    CGGGATCCATATGCTGCTGTTATTAGCAGTTTTTCTGTCGTA42    (2) INFORMATION FOR SEQ ID NO: 20:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 33 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (iii) HYPOTHETICAL: NO    (vii) IMMEDIATE SOURCE:    (B) CLONE: TYROL2    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 20:    GACCGGCCGAAGCTTTAATTAGATGGAGATGGA33    (2) INFORMATION FOR SEQ ID NO: 21:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 25 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (iii) HYPOTHETICAL: NO    (vii) IMMEDIATE SOURCE:    (B) CLONE: Tyrol3    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 21:    AGTGGATCCAGCGGAATGCCGGCTT25    (2) INFORMATION FOR SEQ ID NO: 22:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 17 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (iii) HYPOTHETICAL: NO    (vii) IMMEDIATE SOURCE:    (B) CLONE: TYROL4    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 22:    CGTCATGCTCTTCGGAA17    (2) INFORMATION FOR SEQ ID NO: 23:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 51 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (iii) HYPOTHETICAL: NO    (iii) ANTI-SENSE: NO    (vii) IMMEDIATE SOURCE:    (B) CLONE: TYROL1d    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 23:    CGGGATCCATATGGCTCTGTTATTAGCAGTTTTTCTGTCGTACCTGGCCCG51    (2) INFORMATION FOR SEQ ID NO:24:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 13 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: other nucleic acid    (A) DESCRIPTION: /desc = "oligonucleotide with an EcoRI site"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:    GCAGGAATTCCGG13    __________________________________________________________________________

What is claimed is:
 1. An isolated DNA molecule coding for a cytochromeP-450 monooxygenase that catalyzes the conversion of an amino acid tothe corresponding N-hydroxyamino acid and the conversion of saidN-hydroxyamino acid to the corresponding oxime.
 2. An isolated DNAmolecule according to claim 1, wherein said DNA molecule comprises SEQID NO:
 1. 3. A method of isolating a cDNA molecule coding for acytochrome P-450 monooxygenase that catalyzes the conversion of an aminoacid to the corresponding N-hydroxyamino acid and the conversion of saidN-hydroxyamino acid to the corresponding oxime, comprising:a) isolatingand solubilizing microsomes from plant tissue that produces cyanogenicglycosides or glucosinolates; b) purifying the cytochrome P-450monooxygenase; c) raising antibodies against the purified cytochromeP-450 monooxygenase; d) probing with said antibody a cDNA expressionlibrary of plant tissue that produces cyanogenic glycosides orglucosinolates; and e) isolating clones that express the cytochromeP-450 monooxygenase.
 4. A method of isolating a cDNA molecule coding fora cytochrome P-450 monooxygenase that catalyzes the conversion of anamino acid to the corresponding N-hydroxyamino acid and the conversionof said N-hydroxyamino acid to the corresponding oxime, comprising:a)isolating and solubilizing microsomes from plant tissue that producescyanogenic glycosides or glucosinolates; b) purifying the cytochromeP-450 monooxygenase; c) obtaining a protein sequence of the cytochromeP-450 monooxygenase; d) designing oligonucleotides that specify DNAcoding for 4 to 15 amino acids of said cytochrome P-450 monooxygenaseprotein sequence; e) probing a cDNA library of plant tissue producingcyanogenic glycosides or glucosinolates with said oligonucleotides, orDNA molecules obtained from PCR amplification of cDNA using saidoligonucleotides; and f) isolating clones that encode the cytochromeP-450 monooxygenase.
 5. A method for producing a transgenic plantresistant to insects, acarids, or nematodes, comprising:a) introducinginto a plant cell or plant tissue that can be regenerated into acomplete plant, DNA comprising a gene expressible in said plant thatencodes a cytochrome P-450 monooxygenase that catalyzes the conversionof an amino acid to the corresponding N-hydroxyamino acid and theconversion of said N-hydroxyamino acid to the corresponding oxime; b)selecting transgenic plants; and c) identifying transgenic plants thatare resistant to insects, acarids, or nematodes.
 6. A method forproducing a transgenic plant having improved disease resistance ornutritive value, comprising:a) introducing into a plant cell or planttissue that can be regenerated into a complete plant, DNA encoding senseRNA, antisense RNA, or a ribosome, the expression of which reduces theexpression of a cytochrome P-450 monooxygenase that catalyzes theconversion of an amino acid to the corresponding N-hydroxyamino acid andthe conversion of said N-hydroxyamino acid to the corresponding oxime;b) selecting transgenic plants; and c) identifying transgenic plantshaving improved disease resistance or nutritive value.
 7. An isolatedDNA molecule coding for a cytochrome P-450 monooxygenase that catalyzesthe conversion of an oxime to the corresponding nitrile and theconversion of said nitrile to the corresponding cyanohydrine.
 8. Amethod of isolating a cDNA molecule coding for a cytochrome P-450monooxygenase that catalyzes the conversion of an oxime to thecorresponding nitrile and the conversion of said nitrile to thecorresponding cyanohydrine, comprising:a) isolating and solubilizingmicrosomes from plant tissue that produces cyanogenic glycosides orglucosinolates; b) purifying the cytochrome P-450 monooxygenase; c)raising antibodies against the purified cytochrome P-450 monooxygenase;d) probing with said antibody a cDNA expression library of plant tissuethat produces cyanogenic glycosides or glucosinolates; and e) isolatingclones that express the cytochrome P-450 monooxygenase.
 9. A method ofisolating a cDNA molecule coding for a cytochrome P-450 monooxygenasethat catalyzes the conversion of an oxime to the corresponding nitrileand the conversion of said nitrile to the corresponding cyanohydrine,comprising:a) isolating and solubilizing microsomes from plant tissuethat produces cyanogenic glycosides or glucosinolates; b) purifying thecytochrome P-450 monooxygenase; c) obtaining a protein sequence of thecytochrome P-450 monooxygenase; d) designing oligonucleotides thatspecify DNA coding for 4 to 15 amino acids of said cytochrome P-450monooxygenase protein sequence; e) probing a cDNA library of planttissue producing cyanogenic glycosides or glucosinolates with saidoligonucleotides, or DNA molecules obtained from PCR amplification ofcDNA using said oligonucleotides; and f) isolating clones that encodethe cytochrome P-450 monooxygenase.
 10. A method for producing atransgenic plant resistant to insects, acarids, or nematodes,comprising:a) introducing into a plant cell or plant tissue that can beregenerated into a complete plant, DNA comprising a gene expressible insaid plant that encodes a cytochrome P-450 monooxygenase that catalyzesthe conversion of an oxime to the corresponding nitrile and theconversion of said nitrile to the corresponding cyanohydrine; b)selecting transgenic plants; and c) identifying transgenic plants thatare resistant to insects, acarids, or nematodes.
 11. A method forproducing a transgenic plant having improved disease resistance ornutritive value, comprising:a) introducing into a plant cell or planttissue that can be regenerated into a complete plant, DNA encoding senseRNA, antisense RNA, or a ribosome, the expression of which reduces theexpression of a cytochrome P-450 monooxygenase that catalyzes theconversion of an oxime to the corresponding nitrile and the conversionof said nitrile to the corresponding cyanohydrine; b) selectingtransgenic plants; and c) identifying transgenic plants having improveddisease resistance or nutritive value.
 12. An isolated DNA moleculeaccording to claim 1, wherein said DNA molecule is isolated from a plantthat produces cyanogenic glycosides or glucosinolates.
 13. An isolatedDNA molecule according to claim 1, wherein said DNA molecule is isolatedfrom a plant selected from the group consisting of the genera Sorghum,Trifolium, Linum, Taxus, Triglochin, Mannihot, Amygdalus, Prunus, andcruciferous plants.
 14. An isolated DNA molecule according to claim 13,wherein said DNA molecule is isolated from Sorghum bicolor.
 15. Anisolated DNA molecule according to claim 1, wherein said amino acid isselected from the group consisting of tyrosine, phenylalanine,tryptophan, valine, leucine, isoleucine, and cyclopentenylglycineisoleucine.
 16. An isolated DNA molecule according to claim 15, whereinsaid amino acid is tyrosine.
 17. An isolated DNA molecule according toclaim 1, wherein said cytochrome P-450 monooxygenase has a molecularweight of 57 kD, as determined by SDS-PAGE.
 18. An isolated DNA moleculeaccording to claim 1, wherein said cytochrome P-450 monooxygenasecomprises an amino acid sequence as shown in SEQ ID NO:
 2. 19. Anisolated DNA molecule according to claim 1, wherein said cytochromeP-450 monooxygenase comprises an N-terminal amino acid sequence as shownin SEQ ID NO:
 3. 20. An isolated DNA molecule according to claim 1,wherein said cytochrome P-450 monooxygenase comprises an internal aminoacid sequence selected from the group consisting of SEQ ID NOs: 4-11.21. An isolated DNA molecule according to claim 20, wherein saidcytochrome P-450 monooxygenase comprises an internal amino acid sequenceas shown in SEQ ID NO:
 4. 22. An isolated DNA molecule according toclaim 20, wherein said cytochrome P-450 monooxygenase comprises aninternal amino acid sequence as shown in SEQ ID NO:
 5. 23. An isolatedDNA molecule according to claim 20, wherein said cytochrome P-450monooxygenase comprises an internal amino acid sequence as shown in SEQID NO:
 6. 24. An isolated DNA molecule according to claim 20, whereinsaid cytochrome P-450 monooxygenase comprises an internal amino acidsequence as shown in SEQ ID NO:
 7. 25. An isolated DNA moleculeaccording to claim 20, wherein said cytochrome P-450 monooxygenasecomprises an internal amino acid sequence as shown in SEQ ID NO:
 8. 26.An isolated DNA molecule according to claim 20, wherein said cytochromeP-450 monooxygenase comprises an internal amino acid sequence as shownin SEQ ID NO:
 9. 27. An isolated DNA molecule according to claim 20,wherein said cytochrome P-450 monooxygenase comprises an internal aminoacid sequence as shown in SEQ ID NO:
 10. 28. An isolated DNA moleculeaccording to claim 20, wherein said cytochrome P-450 monooxygenasecomprises an internal amino acid sequence as shown in SEQ ID NO:
 11. 29.An isolated DNA molecule according to claim 7, wherein said DNA moleculeis isolated from a plant that produces cyanogenic glycosides orglucosinolates.
 30. An isolated DNA molecule according to claim 7,wherein said DNA molecule is isolated from a plant selected from thegroup consisting of the genera Sorghum, Trifolium, Linum, Taxus,Triglochin, Mannihot, Amygdalus, Prunus, and cruciferous plants.
 31. Anisolated DNA molecule according to claim 30, wherein said DNA moleculeis isolated from Sorghum bicolor.
 32. An isolated DNA molecule accordingto claim 7, wherein said oxime is obtained by the conversion of an aminoacid to the corresponding N-hydroxyamino acid and the conversion of saidN-hydroxyamino acid to the oxime by another cytochrome P-450monooxygenase.
 33. An isolated DNA molecule according to claim 32,wherein said amino acid is selected from the group consisting oftyrosine, phenylalanine, tryptophan, valine, leucine, isoleucine, andcyclopentenylglycine isoleucine.
 34. An isolated DNA molecule accordingto claim 33, wherein said amino acid is tyrosine.
 35. An isolated DNAmolecule according to claim 7, wherein the ability of said cytochromeP-450 monooxygenase to convert an oxime to the corresponding nitriledepends on the presence of NADPH and wherein this dependency can beovercome by the addition of reductants.
 36. An isolated DNA moleculeaccording to claim 7, wherein said cytochrome P-450 monooxygenase has amolecular weight of 51 kD, as determined by SDS-PAGE.
 37. An isolatedDNA molecule according to claim 7, wherein said cytochrome P-450monooxygenase comprises an N-terminal amino acid sequence as shown inSEQ ID NO:
 12. 38. An isolated DNA molecule according to claim 7,wherein said cytochrome P-450 monooxygenase comprises an amino acidsequence selected from the group consisting of SEQ ID NOs: 13-16.
 39. Anisolated DNA molecule according to claim 38, wherein said cytochromeP-450 monooxygenase comprises an amino acid sequence as shown in SEQ IDNO:
 13. 40. An isolated DNA molecule according to claim 38, wherein saidcytochrome P-450 monooxygenase comprises an amino acid sequence as shownin SEQ ID NO:
 14. 41. An isolated DNA molecule according to claim 38,wherein said cytochrome P-450 monooxygenase comprises an amino acidsequence as shown in SEQ ID NO:
 15. 42. An isolated DNA moleculeaccording to claim 38, wherein said cytochrome P-450 monooxygenasecomprises an amino acid sequence as shown in SEQ ID NO: 16.